Osteogenic devices and methods of use thereof for repair of endochondral bone, osteochondral and chondral defects

ABSTRACT

Disclosed herein are improved osteogenic devices and methods of use thereof for repair of bone and cartilage defects. The devices and methods promote accelerated formation of repair tissue with enhanced stability using less osteogenic protein than devices in the art. Defects susceptible to repair with the instant invention include, but are not limited to: critical size defects, non-critical size defects, non-union fractures, fractures, osteochondral defects, subchondral defects, and detects resulting from degenerative diseases such as osteochondritis dessicans.

CONTINUING APPLICATION DATA

The instant application is a continuing application of co-pending U.S.Ser. No. 08/822,186 filed on Mar. 20, 1997, the entire content of whichis incorporated by reference herein.

FIELD OF THE INVENTION

The invention disclosed herein relates to materials and methods forrepairing bone and cartilage defects using osteogenic proteins.

BACKGROUND OF THE INVENTION

A class of proteins now has been identified that is competent to act astrue chondrogenic tissue morphogens. That is, these proteins are able,on their own, to induce the proliferation and differentiation ofprogenitor cells into functional bone, cartilage, tendon, and/orligamentous tissue. This class of proteins, referred to herein as“osteogenic proteins” or “morphogenic proteins” or “morphogens,”includes members of the family of bone morphogenetic proteins (BMPs)which were initially identified by their ability to induce ectopic,endochondral bone morphogenesis. The osteogenic proteins generally areclassified in the art as a subgroup of the TGF-β superfamily of growthfactors (Hogan (1996) Genes & Development 10:1580-1594). Members of themorphogen family of proteins include the mammalian osteogenic protein-1(OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenicprotein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3),BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog DPP),BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5, BMP-6 and itsmurine homolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF3 (also known asVgr2), GDF8, GDF9, GDF10, GDF11, GDF12, BMP-13, BMP-14, BMP-15, GDF-5(also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7(also known as CDMP-3), the Xenopus homolog Vg1 and NODAL, UNIVIN,SCREW, ADMP, and NEURAL. Members of this family encode secretedpolypeptide chains sharing common structural features, includingprocessing from a precursor “pro-form” to yield a mature polypeptidechain competent to dimerize and containing a carboxy terminal activedomain, of approximately 97-106 amino acids. All members share aconserved pattern of cysteines in this domain and the active form ofthese proteins can be either a disulfide-bonded homodimer of a singlefamily member or a heterodimer of two different members (see, e.g.,Massague (1990) Annu. Rev. Cell Biol, 6:597; Sampath, et al. (1990) J.Biol. Chem. 265:13198). See also, U.S. Pat. No. 5,011,691; U.S. Pat. No.5,266,683, Ozkaynak et al. (1990) EMBO J. 2: 2085-2093, Wharton et al.(1991) PNAS 88:9214-9218), (Ozkaynak (1992) J. Biol. Chem.,267:25220-25227 and U.S. Pat. No. 5,266,683); (Celeste et al. (1991)PNAS 87:9843-9847); (Lyons et al. (1989) PNAS 86:4554-4558). Thesedisclosures describe the amino acid and DNA sequences, as well as thechemical and physical characteristics, of these osteogenic proteins. Seealso, Wozney et al. (1988) Science 242:1528-1534); BMP9 (WO93/00432,published Jan. 7, 1993); DPP (Padgett et al. (1987) Nature 325:81-84;and Vg-1 (Weeks (1987) Cell 51:861-867).

Thus true osteogenic proteins capable of inducing the above-describedcascade of morphogenic events resulting in endochondral bone formation,have now been identified, isolated, and cloned. Whethernaturally-occurring or synthetically prepared, these osteogenic factors,when implanted in a mammal in association with a matrix or substratethat allows attachment, proliferation and differentiation of migratoryprogenitor cells, can induce recruitment of accessible progenitor cellsand stimulate their proliferation, thereby inducing differentiation intochondrocytes and osteoblasts, and further inducing differentiation ofintermediate cartilage, vascularization, bone formation, remodeling,and, finally, marrow differentiation. Furthermore, numerouspractitioners have demonstrated the ability of these osteogenicproteins, when admixed with either naturally-sourced matrix materialssuch as collagen or synthetically-prepared polymeric matrix materials,to induce bone formation, including endochondral bone formation, underconditions where true replacement bone otherwise would not occur. Forexample, when combined with a matrix material, these osteogenic proteinsinduce formation of new bone in large segmental bone defects, spinalfusions, and fractures.

Naturally-sourced matrices, such as collagen, can be replaced with inertmaterials such as plastic, but plastic is not a suitable substitutesince it does not resorb and is limited to applications requiring simplegeometric configurations. To date, biodegradable polymers and copolymershave also been used as matrices admixed with osteogenic proteins forrepair of non-union defects. While such matrices may overcome some ofthe above-described insufficiencies, use of these matrices necessitatesdetermination and control of features such as polymer chemistry,particle size, biocompatibility and other particulars critical foroperability. For example, pores must be formed in the polymer in amanner which ensures adsorption of protein into the matrix andbiodegradation of the matrix. Prior to use of the polymeric matrix,therefore, it is necessary to undergo the extra step of treating thepolymer to induce the formation of pores of the appropriate size.

Standard osteogenic devices, which include either collagen or polymermatrices in admixture with osteogenic protein, lend themselves lessamenable to manipulation during surgery. Standard osteogenic devicesoften have a dry, sandy consistency and can be washed away whenever thedefect site is irrigated during surgery, and/or by blood and/or otherfluids infiltrating the site post-surgery. The addition of certainmaterials to these compositions can aid in providing a more manageablecomposition for handling during surgery. U.S. Pat. Nos. 5,385,887;5,520,923; 5,597,897 and International Publication WO 95/24210 describecompositions containing a synthetic polymer matrix, osteogenic protein,and a carrier for such a purpose. Such compositions have been limited,however, to synthetic polymer matrices because of a desire to overcomecertain alleged adverse immunologic reactions contemplated associatedwith other types of matrices especially biologically-derived matrices,including some forms of collagen. These compositions, therefore, sufferfrom the same feasibility concerns for optimizing polymer chemistry,particle size, biocompatibility, etc., described above.

Needs remain for compositions and methods for repairing bone andcartilage defects which provide greater ease in handling during surgeryand which do not rely on synthetic polymer matrices. Needs also remainfor methods and compositions that can enhance the rate and quality ofnew bone and cartilage formation.

Accordingly, it is an object of the instant invention to provideimproved osteogenic devices and methods of use thereof for repairingbone defects, cartilage defects and/or osteochondral defects that: areeasier to manipulate during surgery; circumvent the concerns of polymerchemistry, particle size and biocompatibility associated with the use ofsynthetic polymer matrices; and, which permit accelerated bone formationand more stable cartilage repair using lower doses of osteogenic proteinthan can be achieved using devices and methods now in the art. It is afurther object of the instant invention to provide osteogenic devicesand methods of use thereof for repairing non-healing, non-union defectsand for promoting articular cartilage repair in chondral orosteochondral defects. Yet another object of the instant invention is toprovide devices and methods for repair of bone and cartilage defectswithout surgical intervention. These and other objects, along withadvantages and features of the invention disclosed herein, will beapparent from the description, drawings and claims that follow.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that admixing osteogenicprotein and a non-synthetic, non-polymeric matrix such as collagen orβ-tricalcium phosphate (β-TCP) with a binding agent yields an improvedosteogenic device with enhanced bone and cartilage repair capabilities.Not only can such improved devices accelerate, the rate of repair, thesedevices also can promote formation of high quality, stable repairtissue, particularly cartilage tissue. Additionally, the foregoingbenefits can be achieved using significantly less osteogenic proteinthan required by standard osteogenic devices. While not wishing to bebound by theory, the aforementioned unexpected properties likely can beattributed to a complementary or synergistic interaction between thenon-polymeric matrix and the binding agent. In view of existingorthopedic and reconstructive technologies, these discoveries areunexpected and were heretofore unappreciated.

The invention provides, in one aspect, a novel device for inducing localbone and cartilage formation comprising osteogenic protein, matrixderived from non-synthetic, non-polymeric material, and binding agent.As contemplated herein, the device preferably comprises osteogenicproteins such as, but not limited to OP-1, OP-2, BMP-2, BMP-4, BMP-5 andBMP-6. A currently preferred osteogenic protein is OP-1. As used herein,the terms “morphogen”, “bone morphogen”, “bone morphogenic protein”,“BMP”, “osteogenic protein” and “osteogenic factor” embrace the class ofproteins typified by human osteogenic protein 1 (hOP-1). Nucleotide andamino acid sequences for hOP-1 are provided in Seq. ID Nos. 1 and 2,respectively. For ease of description, hOP-1 is recited herein below asa representative osteogenic protein. It will be appreciated by theartisan of ordinary skill in the art, however, that OP-1 merely isrepresentative of the TGF-β subclass of true tissue morphogens competentto act as osteogenic proteins, and is not intended to limit thedescription. Other known, and useful proteins include, BMP-2, BMP-3,BMP-3b, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9,GDF-10, GDF-11, GDF-12, NODAL, UNIVIN, SCREW, ADMP, NEURAL andosteogenically active amino acid variants thereof. In one preferredembodiment, the proteins useful in the invention include biologicallyactive species variants of any of these proteins, including conservativeamino acid sequence variants, proteins encoded by degenerate nucleotidesequence variants, and osteogenically active proteins sharing theconserved seven cysteine skeleton as defined herein and encoded by a DNAsequence competent to hybridize to a DNA sequence encoding an osteogenicprotein disclosed herein, including, without limitation, OP-1, BMP-5,BMP-6, BMP-2, BMP-4 or GDF-5, GDF-6 or GDF-7. In another embodiment,useful osteogenic proteins include those sharing the conserved sevencysteine domain and sharing at least 70% amino acid sequence homology(similarity) within the C-terminal active domain, as defined herein. Instill another embodiment, the osteogenic proteins of the invention canbe defined as osteogenically active proteins having any one of thegeneric sequences defined herein, including OPX (SEQ ID No: 3) andGeneric Sequences 7 and 8, or Generic Sequences 9 and 10.

OPX accommodates the homologies between the various species of theosteogenic OP-1 and OP-2 proteins, and is described by the amino acidsequence presented herein below and in SEQ ID NO: 3. Generic sequence 9is a 96 amino acid sequence containing the six cysteine skeleton definedby hOP-1 (residues 335-431 of SEQ ID NO: 2) and wherein the remainingresidues accommodate the homologies of OP-1, OP-2, OP-3, BMP-2, BMP-3,BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-15, GDF-1, GDF-3,GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-11, UNIVIN, NODAL,DORSALIN, NURAL, SCREW and ADMP. That is, each of the non-cysteineresidues is independently selected from the corresponding residue inthis recited group of proteins. Generic Sequence 10 is a 102 amino acidsequence which includes a 5 amino acid sequence added to the N-terminusof the Generic Sequence 9 and defines the seven cysteine skeleton ofhOP-1 (330-431 SEQ ID NO: 2). Generic Sequences 7 and 8 are 96 and 102amino acid sequences, respectively, containing either the six cysteineskeleton (Generic Sequence 7) or the seven cysteine skeleton (GenericSequence 8) defined by hOP-1 and wherein the remaining residuesnon-cysteine accommodate the homologies of: OP-1, OP-2, OP-3, BMP2,BMP3, BMP4, 60A, DPP, Vg1, BMP5, BMP6, Vgr-1, and GDF-1.

As taught below, preferred matrices are non-synthetic, non-polymericmaterials and can be naturally-sourced or derived from biologicalmaterials. Examples of preferred matrices include, but are not limitedto, collagen, demineralized bone and β-TCP. One currently preferredmatrix is collagen. Another currently preferred matrix is β-TCP. Thus,the devices of the instant invention do not comprise as a primarycomponent synthetic polymeric matrices such as homopolymers orcopolymers of α-hydroxy acetic acid and/or α-hydroxy propionic acid,including racemic mixtures thereof.

With respect to binding agents, the instant devices preferably compriseagents useful as gel-forming, viscosity-increasing, suspending and/oremulsifying agents. A currently preferred group of binding agents is thealkycellulose group; especially methylcelluloses such ascarboxymethylcellulose. Other suitable binding agents include othercellulose gums, sodium alginate, dextrans and gelatin powder. Anotherparticularly preferred binding agent is fibrin glue. As used herein theterm “fibrin glue” means a composition comprising mammalian fibrinogenand thrombin. In certain embodiments, the improved devices of theinstant invention further comprise a wetting agent such as, but notlimited to, saline or other aqueous physiological solution.

The improved devices of the instant invention can assume a variety ofconfigurations. The configuration will depend, in part, upon the type ofbinding agent and wetting agent employed. As disclosed herein, onecurrently preferred embodiment can have a putty consistency. Thisparticular configuration is especially suitable for treating opendefects in accordance with the methods of the instant invention. Anothercurrently preferred embodiment of improved osteogenic device can have aviscous fluid consistency. This particular configuration is especiallysuitable for treating closed defects in accordance with the methodsdisclosed herein. Depending upon the configuration of the improveddevice, providing it to a defect site can be accomplished by a varietyof delivery modes. For example, a putty can be packed in and/or aroundthe defect or extruded as a bead from a large-bore apparatus.Alternatively, a viscous liquid can be injected into and/or around thedefect, or alternatively brushed and/or painted on the defect'ssurface(s). Exploitation of a variety of these possible embodiments torepair bone and cartilage defects is exemplified herein.

Among the characteristics of a preferred binding agent is an ability torender the device: pliable, shapeable and/or malleable; injectable;adherent to bone, cartilage, muscle and other tissues; resistant todisintegration upon washing and/or irrigating during surgery; and,resistant to dislodging during surgery, suturing and post-operatively,to name but a few. Additionally, in certain preferred embodiments, abinding agent can achieve the aforementioned features and benefits whenpresent in low proportions. For example, a currently preferred improveddevice comprises approximately 1 part binding agent and approximately 5parts matrix. Certain other preferred embodiments comprise approximately1 part binding agent and approximately 10 parts matrix, while stillothers comprise approximately 1 part binding agent and approximately 25parts matrix. Another currently preferred device comprises approximately3 parts binding agent to 5 parts matrix. Certain binding agents can beused in equal or greater proportions relative to matrix. Anothercurrently preferred device comprises 1 part binding agent and 3 partsmatrix. As exemplified herein, improved devices of widely divergentproportions can induce bone and cartilage formation. Exemplified hereinare improved devices having parts of binding agent to parts of matrixranging from approximately 1:1 to 4:1 as well as from approximately 1:2to 1:5 and 1:10 to 1:25, as well as 1:25 to 1:50. Any proportion ofbinding agent to matrix can be used to practice the instant invention.

Furthermore, the instant invention contemplates that an improvedosteogenic device can comprise more than one matrix material incombination; the relative proportions can be varied to achieve thedesired clinical outcome and can be routinely determined using ordinaryskill. A currently preferred matrix is collagen, especially bovinecollagen. Another suitable matrix is demineralized bone. Yet othersuitable matrices are hydroxyapatites (HAp) of varying calcium:phosphate (Ca/P) molar ratios, porosity and crystallinity; bioactiveceramics; and calcium phosphate ceramics, to name but a few.Additionally, admixtures of the foregoing whereinHAp/tricalciumphosphate ratios are manipulated are also contemplatedherein. In a particularly preferred embodiment, the matrix isβ-tricalcium phosphate (β-TCP).

In another aspect, the instant invention provides methods for inducinglocal bone or cartilage formation for repair of bone, cartilage orosteochondral defects. The instant methods are contemplated as useful toinduce formation of at least endochondral bone, intramembranous bone,and articular cartilage. As disclosed herein, methods of repair includetreatment of both closed and open defects with the above-describedimproved osteogenic devices. As taught herein, the methods of theinstant invention can be practiced using improved devices that are ofsufficient volume to fill the defect site, as well as using improveddevices that are not. Moreover, as a result of this discovery,embodiments are now available for promoting bone and/or cartilage defectrepair without requiring surgical intervention. Availability of suchmethods has implications for compromised individuals such as diabetics,smokers, obese individuals and others whose overall health and impairedblood flow to their extremities are placed at risk when surgicalintervention is required. Examples of defects include, but are notlimited to, critical size defects, non-critical size defects, non-unionfractures, fractures, osteochondral defects, chondral defects andperiodontal defects.

In another aspect, the instant invention provides a kit for practice ofthe above-described methods. As contemplated herein, one embodiment of akit for inducing local bone formation or cartilage formation comprisesan improved device wherein the osteogenic protein and matrix arepackaged in the same receptacle. In other embodiments, the osteogenicprotein, matrix and binding agent are in the same receptacle. In yetother embodiments, wetting agent is also provided and packagedseparately from the other kit components.

Because the instant invention provides practitioners with improvedmaterials and methods for bone and cartilage repair, including repair ofarticular cartilage present in mammalian joints, it overcomes problemsotherwise encountered using the methods and devices of the art. Forexample, the instant invention can induce formation of bona fide hyalinecartilage rather than fibrocartilage at a defect site. Functionalhyaline cartilage forms on the articulating surface of bone at a defectsite and does not degenerate over time to fibrocartilage. By contrast,prior art methods generally ultimately result in development offibrocartilage at the defect site. Unlike hyaline cartilage,fibrocartilage lacks the physiological ability to restore articulatingjoints to their full capacity. Thus, when improved osteogenic devicesare used in accordance with the instant methods, the practitioner cansubstantially restore an osteochondral or a chondral defect in afunctionally articulating joint and avoid the undesirable formation offibrocartilage typical of prior art methods. As contemplated herein, theinvention further embodies allogenic replacement materials for repairingavascular tissue in a skeletal joint which results in formation ofmechanically and functionally viable replacement tissues at a joint.

In summary, the methods, devices, and kits of the present invention canbe used to induce endochondral or intramembranous bone formation forrepairing bone defects which do not heal spontaneously, as well as forpromoting and enhancing the rate and/or quality of new bone formation,particularly in the repair of fractures and fusions, including spinalfusions. The methods, devices, and kits also can induce repair ofosteochondral and/or subchondral defects, i.e., can induce formation ofnew bone and/or the overlying surface cartilage. The present inventionis particularly suitable for use in repair of defects resulting fromdeteriorative or degenerative diseases such as, but not limited to,osteochondritis dessicans. It is also particularly suitable for use inpatients requiring repetitive reconstructive surgeries, as well ascancer patients. Other applications include, but are not limited to,prosthetic repair, spinal fusion, scoliosis, cranial/facial repair, andmassive allograft repair.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects and features of the invention, as wellas the invention itself, may be more fully understood from the followingdescription, when read together with the accompanying drawings, inwhich:

FIG. 1 is a graph depicting cohesiveness properties of varying parts(w/w) of binding agent to parts (w/w) of standard OP device.

FIG. 2 is a graph depicting the effect of varying volumes of wettingagent on the integrity of an improved osteogenic device.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In order to more clearly and concisely describe the subject matter ofthe claimed invention, the following definitions are intended to provideguidance as to the meaning of specific terms used in the followingwritten description and appended claims.

“Bone formation” means formation of endochondral bone or formation ofintramembranous bone. In humans, bone formation begins during the first6-8 weeks of fetal development. Progenitor stem cells of mesenchymalorigin migrate to predetermined sites, where they either: (a) condense,proliferate, and differentiate into bone-forming cells (osteoblasts), aprocess observed in the skull and referred to as “intramembranous boneformation;” or, (b) condense, proliferate and differentiate intocartilage-forming cells (chondroblasts) as intermediates, which aresubsequently replaced with bone-forming cells. More specifically,mesenchymal stem cells differentiate into chondrocytes. The chondrocytesthen become calcified, undergo hypertrophy and are replaced by newlyformed bone made by differentiated osteoblasts, which now are present atthe site. Subsequently, the mineralized bone is extensively remodeled,thereafter becoming occupied by an ossicle filled with functionalbone-marrow elements. This process is observed in long bones andreferred to as “endochondral bone formation.” In postfetal life, bonehas the capacity to repair itself upon injury by mimicking the cellularprocess of embryonic endochondral bone development. That is, mesenchymalprogenitor stem cells from the bone-marrow, periosteum, and muscle canbe induced to migrate to the defect site and begin the cascade of eventsdescribed above. There, they accumulate, proliferate, and differentiateinto cartilage, which is subsequently replaced with newly formed bone.

“Bone” refers to a calcified (mineralized) connective tissue primarilycomprising a composite of deposited calcium and phosphate in the form ofhydroxyapatite, collagen (primarily Type I collagen) and bone cells suchas osteoblasts, osteocytes and osteoclasts, as well as to bone marrowtissue which forms in the interior of true endochondral bone. Bonetissue differs significantly from other tissues, including cartilagetissue. Specifically, bone tissue is vascularized tissue composed ofcells and a biphasic medium comprising a mineralized, inorganiccomponent (primarily hydroxyapatite crystals) and an organic component(primarily of Type I collagen). Glycosaminoglycans constitute less than2% of this organic component and less than 1% of the biphasic mediumitself, or of bone tissue per se. Moreover, relative to cartilagetissue, the collagen present in bone tissue exists in a highly-organizedparallel arrangement. Bony defects, whether from degenerative, traumaticor cancerous etiologies, pose a formidable challenge to thereconstructive surgeon. Particularly difficult is reconstruction orrepair of skeletal parts that comprise part of a multi-tissue complex,such as occurs in mammalian joints.

“Cartilage formation” means formation of connective tissue containingchondrocytes embedded in an extracellular network comprising fibrils ofcollagen (predominantly Type II collagen along with other minor typessuch as Types IX and XI), various proteoglycans, other proteins andwater. “Articular cartilage” refers specifically to hyaline or articularcartilage, an avascular non-mineralized tissue which covers thearticulating surfaces of the portions of bones in joints and allowsmovement in joints without direct bone-to-bone contact, therebypreventing wearing down and damage of opposing bone surfaces. Normalhealthy articular cartilage is referred to as “hyaline,” i.e. having acharacteristic frosted glass appearance. Under physiological conditions,articular cartilage tissue rests on the underlying, mineralized bonesurface called subchondral bone, which contains highly vascularizedossicles. The articular, or hyaline cartilage, found at the end ofarticulating bones is a specialized, histologically distinct tissue andis responsible for the distribution of load resistance to compressiveforces, and the smooth gliding that is part of joint function. Articularcartilage has little or no self-regenerative properties. Thus, if thearticular cartilage is torn or worn down in thickness or is otherwisedamaged as a function of time, disease or trauma, its ability to protectthe underlying bone surface is comprised. In normal articular cartilage,a balance exists between synthesis and destruction of theabove-described extracellular network. However, in tissue subjected torepeated trauma, for example, due to friction between misaligned bonesin contact with one another, or in joint diseases characterized by netloss of articular cartilage, e.g., osteoarthritis, an imbalance occursbetween synthesis and degradation.

Other types of cartilage in skeletal joints include fibrocartilage andelastic cartilage. Secondary cartilaginous joints are formed by discs offibrocartilage that join vertebrae in the vertebral column. Infibrocartilage, the mucopolysaccharide network is interlaced withprominent collagen bundles and the chondrocytes are more widelyscattered than in hyaline cartilage. Elastic cartilage contains collagenfibers that are histologically similar to elastin fibers. Cartilagetissue, including articular cartilage, unlike other connective tissues,lacks blood vessels, nerves, lymphatics and basement membrane. Cartilageis composed of chondrocytes, which synthesize an abundant extracellularmilieu composed of water, collagens, proteoglycans and noncollagenousproteins and lipids. Collagen serves to trap proteoglycans and toprovide tensile strength to the tissue. Type II collagen is thepredominant collagen in cartilage tissue. The proteoglycans are composedof a variable number of glycosaminoglycan chains, keratin sulphate,chondroitin sulphate and/or dermatan sulphate, and N-lined and O-linkedoligosaccharides covalently bound to a protein core.

Articular, or hyaline, cartilage can be distinguished from other formsof cartilage by both its morphology and its biochemistry. Certaincollagens such as the fibrotic cartilaginous tissues, which occur inscar tissue, for example, are keloid and typical of scar-type tissue,i.e., composed of capillaries and abundant, irregular, disorganizedbundles of Type I and Type II collagen. In contrast, articular cartilageis morphologically characterized by superficial versus mid versus deepzones which show a characteristic gradation of features from the surfaceof the tissue to the base of the tissue adjacent to the bone. In thesuperficial zone, for example, chondrocytes are flattened and lieparallel to the surface embedded in an extracellular network thatcontains tangentially arranged collagen and few proteoglycans. In themid zone, chondrocytes are spherical and surrounded by an extracellularnetwork rich in proteoglycans and obliquely organized collagen fibers.In the deep zone, close to the bone, the collage fibers are verticallyoriented. The keratin sulphate rich proteoglycans increase inconcentration with increasing distance from the cartilage surface. For adetailed description of articular cartilage micro-structure, see, forexample, (Aydelotte and Kuettner, (1988), Conn. Tiss. Res. 18:205;Zanetti et al., (1985), J. Cell Biol. 101:53; and Poole et al., (1984),J. Anat. 138:13. Biochemically, articular collagen can be identified bythe presence of Type II and Type IX collagen, as well as by the presenceof well-characterized proteoglycans, and by the absence of Type Xcollagen, which is associated with endochondral bone formation.

Two types of defects are recognized in articular surfaces, i.e.,full-thickness defects and superficial defects. These defects differ notonly in the extent of physical damage to the cartilage, but also in thenature of the repair response each type of lesion can elicit.Full-thickness defects, also referred to herein as “osteochondraldefects,” of an articulating surface include damage to the hyalinecartilage, the calcified cartilage layer and the subchondral bone tissuewith its blood vessels and bone marrow. Full-thickness defects can causesevere pain, since the bone plate contains sensory nerve endings. Suchdefects generally arise from severe trauma and/or during the late stagesof degenerative joint disease, such a osteoarthritis. Full-thicknessdefects may, on occasion, lead to bleeding and the induction of a repairreaction from the subchondral bone. In such instances, however, therepair tissue formed is a vascularized fibrous type of cartilage withinsufficient biomechanical properties, and does not persist on along-term basis. In contrast, superficial defects in the articularcartilage tissue are restricted to the cartilage tissue itself. Suchdefects, also referred to herein as “chondral” or “subchondral defects”,are notorious because they do not heal and show no propensity for repairreactions. Superficial defects may appear as fissures, divots, or cleftsin the surface of the cartilage. They contain no bleeding vessels (bloodspots), such as those seen in full-thickness defects. Superficialdefects may have no known cause, but they are often the result ofmechanical derangements that lead to a wearing down of the cartilaginoustissue. Such mechanical derangements may be caused by trauma to thejoint, e.g., a displacement of torn meniscus tissue into the joint,meniscectomy, a laxation of the joint by a torn ligament, malalignmentof joints, or bone fracture, or by hereditary diseases. Superficialdefects are also characteristic of early stages of degenerative jointdiseases, such as osteoarthritis. Since the cartilage tissue is notinnervated or vascularized, superficial defects do not heal and oftendegenerate into full-thickness defects.

“Defect” or “defect site”, as contemplated herein, can define a bonystructural disruption requiring repair. The defect further can define anosteochondral defect, including a structural disruption of both the boneand overlying cartilage. A defect can assume the configuration of a“void”, which is understood to mean a three-dimensional defect such as,for example, a gap, cavity, hole or other substantial disruption in thestructural integrity of a bone or joint. A defect can be the result ofaccident, disease, surgical manipulation, and/or prosthetic failure. Incertain embodiments, the defect is a void having a volume incapable ofendogenous or spontaneous repair. Such defects are generally twice thediameter of the subject bone and are also called “critical size”defects. For example, in a canine ulna defect model, the art recognizessuch defects to be approximately 3-4 cm, generally at leastapproximately 2.5 cm, gap incapable of spontaneous repair. See, forexample, Schmitz et al., Clinical Orthopaedics and Related Research205:299-308 (1986); and Vukicevic et al., in Advanced in Molecular andCell Biology, Vol. 6, pp. 207-224 (1993)(JAI Press, Inc.), thedisclosures of which are incorporated by reference herein. In rabbit andmonkey segmental defect models, the gap is approximately 1.5 cm and 2.0cm, respectively. In other embodiments, the defect is a non-criticalsize segmental defect. Generally, these are capable of some spontaneousrepair, albeit biomechanically inferior to those made possible bypractice of the instant innovation. In certain other embodiments, thedefect is an osteochondral defect, such as an osteochondral plug. Such adefect traverses the entirety of the overlying cartilage and enters, atleast in part, the underlying bony structure. In contrast, a chondral orsubchondral defect traverses the overlying cartilage, in part or inwhole, respectively, but does not involve the underlying bone. Otherdefects susceptible to repair using the instant invention include, butare not limited to, non-union fractures; bone cavities; tumor resection;fresh fractures (distracted or undistracted); cranial/facialabnormalities; periodontal defects and irregularities; spinal fusions;as well as those defects resulting from diseases such as cancer,arthritis, including osteoarthritis, and other bone degenerativedisorders such as osteochondritis dessicans.

“Repair” is intended to mean new bone and/or cartilage formation whichis sufficient to at least partially fill the void or structuraldiscontinuity at the defect. Repair does not, however, mean, orotherwise necessitate, a process of complete healing or a treatmentwhich is 100% effective at restoring a defect to its pre-defectphysiological/structural/mechanical state.

“Matrix”, as contemplated herein, means a non-polymeric, non-syntheticmaterial that can act as an osteoconductive substrate and has ascaffolding structure on which infiltrating cells can attach,proliferate and participate in the morphogenic process culminating inbone formation. As contemplated herein, matrix does not includepolymeric, synthetic materials such as polymeric matrices comprisinghomopolymers or copolymers of α-hydroxy acetic acid and/or α-hydroxyproponic acid, including racemic mixtures thereof. Specifically,matrices as contemplated herein do not include homopolymers orcopolymers of glycolic acid, lactic acid, and butyric acid, includingderivatives thereof. For example, the matrix of the instant inventioncan be derived from biological, or naturally-sourced, ornaturally-occurring materials. A suitable matrix must be particulate andporous, with porosity being a feature critical to its effectiveness ininducing bone formation, particularly endochondral bone formation. It isunderstood that the term “matrix” means a structural component orsubstrate intrinsically having a three-dimensional form upon whichcertain cellular events involved in endochondral bone morphogenesis willoccur; a matrix acts as a temporary scaffolding structure forinfiltrating cells having interstices for attachment, proliferation anddifferentiation of such cells. The instant invention contemplates thatan improved osteogenic device can comprise more than one matrix materialin combination; the relative proportions can be varied to achieve thedesired clinical outcome and can be routinely determined using ordinaryskill. A currently preferred matrix is collagen, especially bovinecollagen. Another suitable matrix is demineralized bone. Yet othersuitable matrices are hydroxyapatites (HAp) of varying calcium:phosphate (Ca/P) molar ratios, porosity and crystallinity; bioactiveceramics; and calcium phosphate ceramics, to name but a few.Additionally, admixtures of the foregoing whereinHAp/tricalciumphosphate ratios are manipulated are also contemplatedherein. These matrices can be obtained commercially in the form ofgranules, blocks and powders. For example, Pyrost® is a HAp blockderived from bovine bone (Osteo AG, Switzerland); Collapta® is a HApsponge containing collagen (Osteo AG, Switzerland); tricalciumphosphates (β-TCP) can be obtained from Pharma GmbH (Germany) asCerasob®, as well as from Clarkson Chromatography Products, Inc. (S.Williamsport, Pa.) or Osteonics (Netherlands); TCP/HAp granuleadmixtures can be obtained from Osteonics (Netherlands); and 100% HAppowder or granules can be obtained from CAM (a subsidiary of Osteotech,N.J.). Preparation and characterization of certain of the aforementionedmatrices have been extensively described in the art and necessitates nomore than routine experimentation and ordinary skill. See, for example,U.S. Pat. No. 4,975,526; U.S. Pat. No. 5,011,691; U.S. Pat. No.5,171,574; U.S. Pat. No. 5,266,683; U.S. Pat. No. 5,354,557; and U.S.Pat. No. 5,468,845, the disclosures of which are herein incorporated byreference. Other of the aforementioned matrices have also been welldescribed in the art. See, for example, biomaterials treatises such asLeGeros and Daculsi in Handbook of Bioactive Ceramics, II pp. 17-28(1990, CRC Press); and other published descriptions such as Yang Cao,Jie Weng Biomaterials 17, (1996) pp. 419-424; LeGeros, Adv. Dent. Res.2, 164 (1988); Johnson et al., J. Orthopaedic Research, 1996, Vol. 14,pp. 351-369; and Piittelli et al., Biomaterials 1996, Vol. 17, pp.1767-1770, the disclosures of which are herein incorporated byreference.

Sintered, high fired β-TCP (β-tricalcium phosphate) is a currentlypreferred matrix. Sintered β-TCP has a higher dissolution rate thansintered HAps and sintered biphasic calcium phosphate (BCP). The abilityof β-TCP to support bone formation appears to be based, in part, on thesize of the Ca/P granules in the matrix. Sintered β-TCP having particlesizes of between about 212 μm and about 425 μm are most preferred andmay be obtained from Clarkson Chromatography Products, Inc. (S.Williamsport, Pa.) or Osteonics (Netherlands). Upon implantation,devices containing particles sized within this range show high rates ofresorption by image analysis and low inflammatory responses whenimplanted at a rat sub-cutaneous site, as described elsewhere herein.

“Osteogenic device” is understood to mean a composition comprising atleast osteogenic protein dispersed in a matrix. As disclosed herein, an“improved osteogenic device” comprises osteogenic protein, a matrix asdefined above, and a binding agent as defined below. In contrast, a“standard osteogenic device” comprises osteogenic protein and a matrix,but not a binding agent; standard osteogenic devices can comprise eithera synthetic, polymeric or a matrix as defined above. In the Examples andteachings set forth below, standard osteogenic devices are furtherdesignated; standard devices, OP device, OP-1 device, or OP. Improvedosteogenic devices are further designated: CMC-containing device,CMC-containing standard device, CMC/OP-1 device, OP-1/CMC/collagen,OPCMC/collagen, and fibrin glue-containing improved device. As usedherein, a “mock device” does not contain osteogenic protein and isformulated free of any known osteoinductive factor. The instantinvention also contemplates improved devices comprising at least twodifferent osteogenic proteins and/or at least two different matrices; asdefined herein. Other embodiments of improved device can furthercomprise at least two different binding agents, as defined herein. Instill other embodiments, any one of the aforementioned improved devicescan further comprise a wetting agent, as defined herein. Any of theaforementioned embodiments can also include radiopaque components, suchas commercially available contrast agents. Generally, there are threewell-known types of such agents—hydroxyapatites, barium sulfate, andorganic iodine. Devices containing radiopaque components areparticularly useful for device administration at a closed defect site,as discussed elsewhere herein. Identification of a suitable radiopaquecomponent requires only ordinary skill and routine experimentation. See,for example, radiographic treatises including, Ehrlich and McCloskey,Patient Care in Radiography (Mosby Publisher, 1993); Carol, Fuch'sRadiographic Exposure, Processing and Quality Control (Charles C. ThomasPublisher, 1993); and Snopek, Fundamentals of Special RadiographicProcedures, (W.B. Saunders Company, 1992), the disclosures of which areherein incorporated by reference.

Preferred embodiments of improved devices are adherent to bone,cartilage, muscle and/or other tissue. They have improved handlingproperties and are resistant to dislodging upon irrigation duringsurgery and upon suturing. Similarly, they are cohesive and not washedaway, disintegrated or diluted by irrigation and/or infiltrating bodyfluids such as blood. Preferred embodiments remain adherentpost-surgery, even at an articulating joint. Of particular importance isthat improved devices are readily confined to the defect site.Functionally, the improved osteogenic device of the instant inventioninduces accelerated bone and/or cartilage formation, as well as higherquality, more stable repair tissue and can achieve those benefits atdoses of osteogenic protein lower than required with a standardosteogenic device. Thus, the admixture of osteogenic protein withnon-synthetic, non-polymeric matrix and a binding agent has unexpectedproperties upon which the skilled practitioner can now capitalize asexemplified herein. One currently preferred embodiment comprises OP-1,collagen matrix and the binding agent carboxymethylcellulose (CMC). Asdiscussed below, an advantage associated with the binding agent, CMC, isits effectiveness even when present in low relative amounts. Forexample, in certain embodiments exemplified herein, OP-1 can be used inamounts ranging from approximately 1.25 to 2.50 mg per approximately1000 mg collagen and per approximately 180 to 200 mg CMC. Othercurrently preferred embodiments comprise OP-1, collagen matrix and thebinding agent fibrin glue; or OP-1, β-TCP matrix and the binding agentfibrin glue. In certain embodiments exemplified herein, approximately 40mg fibrin glue can be used with 1000 mg β-TCP or 1000 mg collagen. Inyet other embodiments, approximately 20 mg fibrin glue can be used with1000 mg collagen to support bone and/or cartilage formation. Thesematrices and binding agents exhibit all of the aforementioned preferredhandling characteristics associated with an improved osteogenic device.

In certain other embodiments, these amounts of protein, matrix andbinding agent can be increased or decreased according to the conditionsand circumstances related to defect repair. A wetting agent such assaline can be further added. As exemplified below, a preferredconfiguration for implantation at an open defect site assumes a puttyconsistency. It can be molded and shaped by the surgeon prior toimplantation. This configuration is achieved by adjusting the proportionof matrix to binding agent to wetting agent in a manner similar to thattaught herein. As further exemplified below, closed defects can betreated with a looser, more fluid device configuration resembling aviscous liquid. Such configurations can be injected without surgicalintervention at a defect site. Again, merely adjusting the proportionsof matrix to binding agent to wetting agent can achieve this embodiment.Currently, a preferred improved device comprises approximately 1 partbinding agent (w/w) to approximately 5 parts matrix (w/w). As describedherein below, other proportions can be used to prepare improved devices,depending upon the nature of binding agent and/or matrix.

Of course, an essential feature of any formulation of improvedosteogenic device is that it must be effective to provide at least alocal source of osteogenic protein at the defect site, even iftransient. As exemplified below, the binding agent content of animproved osteogenic device does not affect protein release/retentionkinetics. This is unexpected in view of contrary observations thatpolymer-containing standard devices failed to show clinicallysignificant osteoinducing effects in the absence of sequesteringmaterial (defined to include cellulosic materials) because proteindesorbtion was too great. (See, for example, U.S. Pat. No. 5,597,897.)As exemplified below, even when a binding agent as defined herein ispresent, protein is still desorbed from the improved device yetosteoinductive effects are readily apparent. While not wishing to bebound by theory, the unexpected features and benefits associated withthe instant invention appear to relate less to a protein-binding agentinteraction and more to a binding agent-matrix interaction.Specifically, binding agents as defined herein appear to complementand/or interact synergistically with the matrix required by the instantinvention. This has heretofore been unappreciated, and this combinationis discouraged by the teachings of the prior art. (See, for example,U.S. Pat. Nos. 5,520,923; 5,597,897; and WO 95/24210.)

The term “unitary” device refers to an improved osteogenic deviceprovided to the practitioner as a single, pre-mixed formulationcomprising osteogenic protein, matrix and binding agent. The term“non-unitary” device refers to an improved osteogenic device provided tothe practitioner in at least two separate packages for admixing prior touse. Typically, a non-unitary device comprises at least binding agentpackaged separately from the osteogenic protein and the matrix. The term“carrier” refers to an admixture of binding agent and matrix, as each isdefined herein. Thus, for example, an improved osteogenic device asdisclosed herein comprises osteogenic protein and a carrier.

In addition to osteogenic proteins, various growth factors, hormones,enzymes, therapeutic compositions, antibiotics, or other bioactiveagents can also be contained within an improved osteogenic device. Thus,various known growth factors such as EGF, PDGF, IGF, FGF, TGF-α, andTGF-β can be combined with an improved osteogenic device and deliveredto the defect site. An improved osteogenic device can also be used todeliver chemotherapeutic agents, insulin, enzymes, enzyme inhibitorsand/or chemoattractant/chemotactic factors.

“Osteogenic protein”, or bone morphogenic protein, is generallyunderstood to mean a protein which can induce the full cascade ofmorphogenic events culminating in endochondral bone formation. Asdescribed elsewhere herein, the class of proteins is typified by humanosteogenic protein (hOP-1). Other osteogenic proteins useful in thepractice of the invention include osteogenically active forms of OP-1,OP-2, OP-3, BMP2, BMP3, BMP4, BMP5, BMP6, BMP9, DPP, Vg1, Vgr, 60Aprotein, GDF-1, GDF-3, GDF-5, 6, 7, BMP10, BMP11, BMP13, BMP15, UNIVIN,NODAL, SCREW, ADMP or NEURAL and amino acid sequence variants thereof.In one currently preferred embodiment, osteogenic protein includes anyone of: OP-1, OP-2, OP-3, BMP2, BMP4, BMP5, BMP6, BMP9, and amino acidsequence variants and homologs thereof, including species homologsthereof. Particularly preferred osteogenic proteins are those comprisingan amino acid sequence having at least 70% homology with the C-terminal102-106 amino acids, defining the conserved seven cysteine domain, ofhuman OP-1, BMP2, and related proteins. Certain preferred embodiments ofthe instant invention comprise the osteogenic protein, OP-1. Certainother preferred embodiments comprise mature OP-1 solubilized in aphysiological saline solution. As further described elsewhere herein,the osteogenic proteins suitable for use with Applicants' invention canbe identified by means of routine experimentation using theart-recognized bioassay described by Reddi and Sampath. “Amino acidsequence homology” is understood herein to mean amino acid sequencesimilarity. Homologous sequences share identical or similar amino acidresidues, where similar residues are conservative substitutions for, orallowed point mutations of, corresponding amino acid residues in analigned reference sequence. Thus, a candidate polypeptide sequence thatshares 70% amino acid homology with a reference sequence is one in whichany 70% of the aligned residues are either identical to, or areconservative substitutions of, the corresponding residues in a referencesequence. Examples of conservative variations include the substitutionof one hydrophobic residue, such as isoleucine, valine, leucine ormethionine, for another, or the substitution of one polar residue foranother, such as the substitution of arginine for lysine, glutamic foraspartic acids, or glutamine for asparagine, and the like. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid, provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

Proteins useful in this invention include eukaryotic proteins identifiedas osteogenic proteins (see U.S. Pat. No. 5,011,691, incorporated hereinby reference), such as the OP-1, OP-2, OP-3 and CBMP-2 proteins, as wellas amino acid sequence-related proteins, such as DPP (from Drosophila),Vg1 (from Xenopus), Vgr-1 (from mouse), GDF-1 (from humans, see Lee(1991), PNAS 88:4250-4254), 60A (from Drosophila, see Wharton et al.(1991) PNAS 88:9214-9218), dorsalin-1 (from chick, see Basler et al.(1993) Cell 73:687-702 and GenBank accession number L12032) and GDF-5(from mouse, see Storm et al. (1994) Nature 368:639-643). BMP-3 is alsopreferred. Additional useful proteins include biosynthetic morphogenicconstructs disclosed in U.S. Pat. No. 5,011,691, e.g., COP-1,3-5, 7 and16, as well as other proteins known in the art. Still other proteinsinclude osteogenically active forms of BMP-3b (see Takao, et al.,(1996), Biochem. Biophys. Res. Comm. 219: 656-662. BMP-9 (seeWO95/33830), BMP-15 (see WO96/35710), BMP-12 (see WO95/16035), CDMP-1(see WO 94/12814), CDMP-2 (see WO94/12814), BMP-10 (see WO94/26893),GDF-1 (see WO92/00382), GDF-10 (see WO95/10539), GDF-3 (see WO94/15965)and GDF-7 (WO95/01802).

Still other useful proteins include proteins encoded by DNAs competentto hybridize to a DNA encoding an osteogenic protein as describedherein, and related analogs, homologs, muteins (biosynthetic variants)and the like (see below). Certain embodiments of the improved osteogenicdevices contemplated herein comprise osteogenic protein functionallyand/or stably linked to matrix.

“Binding Agent”, as used herein, means any physiologically-compatiblematerial which, when admixed with osteogenic protein and matrix asdefined herein, promotes bone and/or cartilage formation. Certainpreferred binding agents promote such repair using less osteogenicprotein than standard osteogenic devices. Other preferred binding agentscan promote repair using the same amount of the osteogenic protein thanthe standard osteogenic devices while some require more to promoterepair. As taught herein, the skilled artisan can determine an effectiveamount of protein for use with any suitable binding agent using onlyroutine experimentation. Among the other characteristics of a preferredbinding agent is an ability to render the device: pliable, shapeableand/or malleable; injectable; adherent to bone, cartilage, muscle andother tissues; resistant to disintegration upon washing and/orirrigating during surgery; and, resistant to dislodging during surgery,suturing and post-operatively, to name but a few. Additionally, incertain preferred embodiments, a binding agent can achieve theaforementioned features and benefits when present in low proportions.For example, a currently preferred improved device comprisesapproximately 1 part binding agent and approximately 5 parts matrix.Another currently preferred device comprises approximately 3 partsbinding agent to 5 parts matrix. Yet other preferred devices compriseapproximately 1 part binding agent and approximately 10 parts matrixwhile others comprise approximately 1 part binding agent andapproximately 25 or 50 parts matrix. Certain binding agents can be usedin equal or greater proportions relative to matrix, but, such agentsshould be tested as taught below to identify possible matrix dilutioneffects.

Those binding agents contemplated as useful herein include, but are notlimited to: art-recognized suspending agents, viscosity-producing agentsand emulsifying agents. In particular, art-recognized agents, such ascellulose gum derivatives, sodium alginate, and gelatin powder can beused. More particularly, cellulosic agents such as alkylcelluloses, arepreferred including agents such as methylcellulose,methylhydroxyethylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose, and hydroxyalkylcelluloses, to name but a few.Currently, among the most preferred is carboxymethylcellulose, includingthe sodium salt thereof. As exemplified below, other binding agentssuitable for use in the instant invention include, but are not limitedto, dextran, mannitol, white petrolatum, sesame oil and admixturesthereof. Finally, also among the most preferred binding agents is fibringlue, which comprises a mixture of mammalian fibrinogen and thrombin. Inview of the teachings set forth herein, the artisan can identifysuitable equivalents of the above-identified binding agents using merelyroutine experimentation and ordinary skill.

“Wetting Agent”, as used herein, means any physiologically-compatibleaqueous solution, provided it does not interfere with bone and/orcartilage formation. In certain embodiments of the instant invention,wetting agent is admixed with an improved device to achieve theconsistency necessitated by the mode of defect repair. As taught herein,wetting agent can be used to achieve a putty configuration or,alternatively, a viscous liquid configuration. A currently preferredwetting agent is physiological saline. Equivalents can be identified bythe artisan using no more than routine experimentation and ordinaryskill.

The means for making and using the methods, implants and devices of theinvention, as well as other material aspects concerning their nature andutility, including how to make and how to use the subject matterclaimed, will be further understood from the following, whichconstitutes the best mode currently contemplated for practicing theinvention. It will be appreciated that the invention is not limited tosuch exemplary work or to the specific details set forth in theseexamples.

I. Protein Considerations A. Biochemical, Structural and FunctionalProperties of Bone Morphogenic Proteins

Naturally occurring proteins identified and/or appreciated herein to beosteogenic or bone morphogenic proteins form a distinct subgroup withinthe loose evolutionary grouping of sequence-related proteins known asthe TGF-β superfamily or supergene family. The naturally occurring bonemorphogens share substantial amino acid sequence homology in theirC-terminal regions (domains). Typically, the above-mentioned naturallyoccurring osteogenic proteins are translated as a precursor, having anN-terminal signal peptide sequence typically less than about 30residues, followed by a “pro” domain that is cleaved to yield the matureC-terminal domain. The signal peptide is cleaved rapidly upontranslation, at a cleavage site that can be predicted in a givensequence using the method of Von Heijne (1986) Nucleic Acids Research14:4683-4691. The pro domain typically is about three times larger thanthe fully processed mature C-terminal domain.

In preferred embodiments, the pair of morphogenic polypeptides haveamino acid sequences each comprising a sequence that shares a definedrelationship with an amino acid sequence of a reference morphogen.Herein, preferred osteogenic polypeptides share a defined relationshipwith a sequence present in osteogenically active human OP-1, SEQ ID NO:2. However, any one or more of the naturally occurring or biosyntheticsequences disclosed herein similarly could be used as a referencesequence. Preferred osteogenic polypeptides share a defined relationshipwith at least the C-terminal six cysteine domain of human OP-1, residues335-431 of SEQ ID NO: 2. Preferably, osteogenic polypeptides share adefined relationship with at least the C-terminal seven cysteine domainof human OP-1, residues 330-431 of SEQ ID NO: 2. That is, preferredpolypeptides in a dimeric protein with bone morphogenic activity eachcomprise a sequence that corresponds to a reference sequence or isfunctionally equivalent thereto.

Functionally equivalent sequences include functionally equivalentarrangements of cysteine residues disposed within the referencesequence, including amino acid insertions or deletions which alter thelinear arrangement of these cysteines, but do not materially impairtheir relationship in the folded structure of the dimeric morphogenprotein, including their ability to form such intra- or inter-chaindisulfide bonds as may be necessary for morphogenic activity.Functionally equivalent sequences further include those wherein one ormore amino acid residues differs from the corresponding residue of areference sequence, e.g., the C-terminal seven cysteine domain (alsoreferred to herein as the conserved seven cysteine skeleton) of humanOP-1, provided that this difference does not destroy bone morphogenicactivity. Accordingly, conservative substitutions of corresponding aminoacids in the reference sequence are preferred. Amino acid residues thatare conservative substitutions for corresponding residues in a referencesequence are those that are physically or functionally similar to thecorresponding reference residues, e.g., that have similar size, shape,electric charge, chemical properties including the ability to formcovalent or hydrogen bonds, or the like. Particularly preferredconservative substitutions are those fulfilling the criteria defined foran accepted point mutation in Dayhoff et al. (1978), 5 Atlas of ProteinSequence and Structure, Suppl. 3, ch. 22 (pp. 354-352), Natl. Biomed.Res. Found., Washington, D.C. 20007, the teachings of which areincorporated by reference herein.

Examples of conservative substitutions include: Conservativesubstitutions typically include the substitution of one amino acid foranother with similar characteristics, e.g., substitutions within thefollowing groups: valine, glycine; glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. The term“conservative variation” also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid provided thatantibodies raised to the substituted polypeptide also immunoreact withthe unsubstituted polypeptide.

Natural-sourced osteogenic protein in its mature, native form is aglycosylated dimer typically having an apparent molecular weight ofabout 30-36 kDa as determined by SDS-PAGE. When reduced, the 30 kDaprotein gives rise to two glycosylated peptide subunits having apparentmolecular weights of about 16 kDa and 18 kDa. In the reduced state, theprotein has no detectable osteogenic activity. The unglycosylatedprotein, which also has osteogenic activity, has an apparent molecularweight of about 27 kDa. When reduced, the 27 kDa protein gives rise totwo unglycosylated polypeptides, having molecular weights of about 14kDa to 16 kDa, capable of inducing endochondral bone formation in amammal. As described above, particularly useful sequences include thosecomprising the C-terminal 96 or 102 amino acid sequences of DPP (fromDrosophila), Vg1 (from Xenopus), Vgr-1 (from mouse), the OP-1 and OP-2proteins, proteins (see U.S. Pat. No. 5,011,691 and Oppermann et al., aswell as the proteins referred to as BMP2, BMP3, BMP4 (see WO88/00205,U.S. Pat. No. 5,013,649 and WO91/18098), BMP5 and BMP6 (see WO90/11366,PCT/US90/01630), BMP8 and BMP9.

Other morphogenic proteins useful in the practice of the inventioninclude morphogenically active forms of OP-1, OP-2, OP-3, BMP2, BMP3,BMP4, BMP5, BMP6, BMP9, GDF-5, GDF-6, GDF-7, DPP, Vg1, Vgr, 60A protein,GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, BMP10, BMP11, BMP13, BMP15, UNIVIN,NODAL, SCREW, ADMP or NURAL and amino acid sequence variants thereof. Inone currently preferred embodiment, osteogenic protein include any oneof: OP-1, OP-2, OP-3, BMP2, BMP4, BMP5, BMP6, BMP9, and amino acidsequence variants and homologs thereof, including species homologs,thereof.

Publications disclosing these sequences, as well as their chemical andphysical properties, include: OP-1 and OP-2: U.S. Pat. No. 5,011,691,U.S. Pat. No. 5,266,683, Ozkaynak et al. (1990) EMBO J. 9: 2085-2093;OP-3: WO94/10203 (PCT US93/10520); BMP2, BMP3, BMP4: WO88/00205, Wozneyet al. (1988) Science 242: 1528-1534); BMP5 and BMP6: Celeste et al.(1991) PNAS 87: 9843-9847; Vgr-1: Lyons et al. (1989) PNAS 86:4554-4558; DPP: Padgett et al. (1987) Nature 325: 81-84; Vg-1: Weeks(1987) Cell 51: 861-867; BMP-9: WO95/33830 (PCT/US95/07084); BMP10:WO94/26893 (PCT/US94/05290); BMP-11: WO94/26892 (PCT/US94/05288); BMP12:WO95/16035 (PCT/US94/14030); BMP-13: WO95/16035 (PCT/US94/14030); GDF-1:WO92/00382 (PCT/US91/04096) and Lee et al. (1991) PNAS 88: 4250-4254;GDF-8: WO94/21681 (PCT/US94/03019); GDF-9: WO94/15966 (PCT/US94/00685);GDF-10: WO95/10539 (PCT/US94/11440); GDF-11: WO96/01845(PCT/US95/08543); BMP-15: WO96/36710 (PCT/US96/06540); MP121: WO96/01316(PCT/EP95/02552); GDF-5 (CDMP-1, MP52): WO94/15949 (PCT/US94/00657) andWO96/14335 (PCT/US94/12814) and WO93/16099 (PCT/EP93/00350); GDF-6(CDMP-2, BMP13): WO95/01801 (PCT/US94/07762) and WO96/14335 andWO95/10635 (PCT/US94/14030); GDF-7 (CDMP-3, BMP12): WO95/10802(PCT/US94/07799) and WO95/10635 (PCT/US94/14030). In another embodiment,useful proteins include biologically active biosynthetic constructs,including novel biosynthetic morphogenic proteins and chimeric proteinsdesigned using sequences from two or more known morphogens. See also thebiosynthetic constructs disclosed in U.S. Pat. No. 5,011,691, thedisclosure of which is incorporated herein by reference (e.g., COP-1,COP-3, COP-4, COP-5, COP-7, and COP-16).

In certain preferred embodiments, bone morphogenic proteins usefulherein include those in which the amino acid sequences comprise asequence sharing at least 70% amino acid sequence homology or“similarity”, and preferably 80% homology or similarity, with areference morphogenic protein selected from the foregoing naturallyoccurring proteins. Preferably, the reference protein is human OP-1, andthe reference sequence thereof is the C-terminal seven cysteine domainpresent in osteogenically active forms of human OP-1, residues 330-431of SEQ ID NO: 2. In certain embodiments, a polypeptide suspected ofbeing functionally equivalent to a reference morphogen polypeptide isaligned therewith using the method of Needleman, et al. (1970) J. Mol.Biol. 48:443-453, implemented conveniently by computer programs such asthe Align program (DNAstar, Inc.). As noted above, internal gaps andamino acid insertions in the candidate sequence are ignored for purposesof calculating the defined relationship, conventionally expressed as alevel of amino acid sequence homology or identity, between the candidateand reference sequences. “Amino acid sequence homology” is understoodherein to include both amino acid sequence identity and similarity.Homologous sequences share identical and/or similar amino acid residues,where similar residues are conservation substitutions for, or “allowedpoint mutations” of, corresponding amino acid residues in an alignedreference sequence. Thus, a candidate polypeptide sequence that shares70% amino acid homology with a reference sequence is one in which any70% of the aligned residues are either identical to, or are conservativesubstitutions of, the corresponding residues in a reference sequence. Ina currently preferred embodiment, the reference sequence is OP-1. Bonemorphogenic proteins useful herein accordingly include allelic,phylogenetic counterpart and other variants of the preferred referencesequence, whether naturally-occurring or biosynthetically produced(e.g., including “muteins” or “mutant proteins”), as well as novelmembers of the general morphogenic family of proteins, including thoseset forth and identified above. Certain particularly preferredmorphogenic polypeptides share at least 60% amino acid identity with thepreferred reference sequence of human OP-1, still more preferably atleast 65% amino acid identity therewith.

In other preferred embodiments, the family of bone morphogenicpolypeptides useful in the present invention, and members thereof, aredefined by a generic amino acid sequence. For example, Generic Sequence7 (SEQ ID NO: 4) and Generic Sequence 8 (SEQ ID NO: 5) disclosed below,accommodate the homologies shared among preferred protein family membersidentified to date, including at least OP-1, OP-2, OP-3, CBMP-2A,CBMP-2B, BMP-3, 60A, DPP, Vg1, BMP-5, BMP-6, Vgr-1, and GDF-1. The aminoacid sequences for these proteins are described herein and/or in theart, as summarized above. The generic sequences include both the aminoacid identity shared by these sequences in the C-terminal domain,defined by the six and seven cysteine skeletons (Generic Sequences 7 and8, respectively), as well as alternative residues for the variablepositions within the sequence. The generic sequences provide anappropriate cysteine skeleton where inter- or intramolecular disulfidebonds can form, and contain certain critical amino acids likely toinfluence the tertiary structure of the folded proteins. In addition,the generic sequences allow for an additional cysteine at position 36(Generic Sequence 7) or position 41 (Generic Sequence 8), therebyencompassing the morphogenically active sequences of OP-2 and OP-3.

Generic Sequence 7             Leu Xaa Xaa Xaa Phe Xaa Xaa              1               5 Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa Pro         10                  15 Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly         20                  25 Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa         30                  35 Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa         40                  45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa         50                  55 Xaa Xaa Xaa Cys Cys Xaa Pro Xaa Xaa Xaa         60                  65 Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa         70                  75 Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa         80                  85 Xaa Met Xaa Val Xaa Xaa Cys Xaa Cys Xaa         90                  95wherein each Xaa independently is selected from a group of one or morespecified amino acids defined as follows: “Res.” means “residue” and Xaaat res. 2=(Tyr or Lys); Xaa at res. 3=Val or Ile); Xaa at res. 4=(Ser,Asp or Glu); Xaa at res. 6=(Arg, Gln, Ser, Lys or Ala); Xaa at res.7=(Asp or Glu); Xaa at res. 8=(Leu, Val or Ile); Xaa at res. 11=(Gln,Leu, Asp, His, Asn or Ser); Xaa at res. 12=(Asp, Arg, Asn or Glu); Xaaat res. 13=(Trp or Ser); Xaa at res. 14=(Ile or Val); Xaa at res.15=(Ile or Val); Xaa at res. 16 (Ala or Ser); Xaa at res. 18=(Glu., Gln,Leu, Lys, Pro or Arg); Xaa at res. 19=(Gly or Ser); Xaa at res. 20=(Tyror Phe); Xaa at res. 21=(Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaaat res. 23=(Tyr, Asn or Phe); Xaa at res. 26=(Glu, His, Tyr, Asp, Gln,Ala or Ser); Xaa at res. 28=(Glu, Lys, Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro, Gln, Ile or Asn); Xaa at res. 31=(Phe, Leu or Tyr);Xaa at res. 33=(Leu, Val or Met); Xaa at res. 34 (Asn, Asp, Ala, Thr orPro); Xaa at res. 35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res.36=(Tyr, Cys, His, Ser or Ile); Xaa at res. 37=(Met, Phe, Gly or Leu);Xaa at res. 38=(Asn, Ser or Lys); Xaa at res. 39=(Ala, Ser, Gly or Pro);Xaa at res. 40=(Thr, Leu or Ser); Xaa at res. 44=(Ile, Val or Thr); Xaaat res. 45=(Val, Leu, Met or Ile); Xaa at res. 46=(Gln or Arg); Xaa atres. 47=(Thr, Ala or Ser); Xaa at res. 48=(Leu or Ile); Xaa at res.49=(Val or Met); Xaa at res. 50=(His, Asn or Arg); Xaa at res. 51=(Phe,Leu, Asn, Ser, Ala or Val); Xaa at res. 52=(Ile, Met, Asn, Ala, Val, Glyor Leu); Xaa at res. 53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or Val); Xaa at res. 55=(Glu, Asp, Asn, Gly, Val, Pro orLys); Xaa at res. 56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile orHis); Xaa at res. 57=(Val, Ala or Ile); Xaa at res. 58=(Pro or Asp); Xaaat res. 59=(Lys, Leu or Glu); Xaa at res. 60=(Pro, Val or Ala); Xaa atres. 63=(Ala or Val); Xaa at res. 65=(Thr, Ala or Glu); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res. 67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser, Asp or Gly); Xaa at res. 69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res. 71=(Ser, Ala or Pro); Xaa at res.72=(Val, Leu, Met or Ile); Xaa at res. 74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res. 76=(Asp, Asn or Leu); Xaa at res.77=(Asp, Glu, Asn, Arg or Ser); Xaa at res. 78=(Ser, Gln, Asn, Tyr orAsp); Xaa at res. 79=(Ser, Mn, Asp, Glu or Lys); Xaa at res. 80=(Asn,Thr or Lys); Xaa at res. 82=(Ile, Val or Asn); Xaa at res. 84=

(Lys or Arg); Xaa at res. 85=(Lys, Asn, Gln, His, Arg or Val); Xaa atres. 86=(Tyr, Glu or His); Xaa at res. 87=(Arg, Gln, Glu or Pro); Xaa atres. 88=(Asn, Glu, Trp or Asp); Xaa at res. 90=(Val, Thr, Ala or Ile);Xaa at res. 92=(Arg, Lys, Val, Asp, Gln or Glu); Xaa at res. 93=(Ala,Gly, Glu or Ser); Xaa at res. 95=(Gly or Ala) and Xaa at res. 97=(His orArg).

Generic Sequence 8 (SEQ ID NO: 5) includes all of Generic Sequence 7 andin addition includes the following sequence (SEQ ID NO: 8) at itsN-terminus:

Cys Xaa Xaa Xaa Xaa   1               5Accordingly, beginning with residue 7, each “Xaa” in Generic Sequence 8is a specified amino acid defined as for Generic Sequence 7, with thedistinction that each residue number described for Generic Sequence 7 isshifted by five in Generic Sequence 8. Thus, “Xaa at res. 2=(Tyr orLys)” in Generic Sequence 7 refers to Xaa at res. 7 in Generic Sequence8. In Generic Sequence 8, Xaa at res. 2=(Lys, Arg, Ala or Gln); Xaa atres. 3=(Lys, Arg or Met); Xaa at res. 4=(His, Arg or Gln); and Xaa atres. 5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or Tyr).

In another embodiment, useful osteogenic proteins include those definedby Generic Sequences 9 and 10, defined as follows.

Specifically, Generic Sequences 9 and 10 are composite amino acidsequences of the following proteins: human OP-1, human OP-2, human OP-3,human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, humanBMP-8, human BMP-9, human BMP10, human BMP-11, Drosophila 60A, XenopusVg-1, sea urchin UNIVIN, human CDMP-1 (mouse GDF-5), human CDMP-2 (mouseGDF-6, human BMP-13), human CDMP-3 (mouse GDF-7, human BMP-12), mouseGDF-3, human GDF-1, mouse GDF-1, chicken DORSALIN, dpp, DrosophilaSCREW, mouse NODAL, mouse GDF-8, human GDF-8, mouse GDF-9, mouse GDF-10,human GDF-11, mouse GDF-11, human BMP-15, and rat BMP3b. Like GenericSequence 7, Generic Sequence 9 accommodates the C-terminal six cysteineskeleton and, like Generic Sequence 8, Generic Sequence 10 accommodatesthe seven cysteine skeleton.

Generic Sequence 9 (SEQ ID NO: 6)Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1               5                   10Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa                15                  20Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa                25                  30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                35                  40Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                45                  50Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                55                  60Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa                65                  70Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa                75                  80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa                85                  90 Xaa Xaa Xaa Cys Xaa Cys Xaa                95wherein each Xaa is independently selected from a group of one or morespecified amino acids defined as follows: “Res.” means “residue” and Xaaat res. 1=(Phe, Leu or Glu); Xaa at res. 2=(Tyr, Phe, His, Arg, Thr,Lys, Gln, Val or Glu); Xaa at res. 3=(Val, Ile, Leu or Asp); Xaa at res.4=(Ser, Asp, Glu, Asn or Phe); Xaa at res. 5=(Phe or Glu); Xaa at res.6=(Arg, Gln, Lys, Ser, Glu, Ala or Asn); Xaa at res. 7=(Asp, Glu, Leu,Ala or Gln); Xaa at res. 8=(Leu, Val, Met, Ile or Phe); Xaa at res.9=(Gly, His or Lys); Xaa at res. 10=(Trp or Met); Xaa at res. 11=(Gln,Leu, His, Glu, Asn, Asp, Ser or Gly); Xaa at res. 12=(Asp, Asn, Ser,Lys, Arg, Glu or His); Xaa at res. 13=(Trp or Ser); Xaa at res. 14=(Ileor Val); Xaa at res. 15=(Ile or Val); Xaa at res. 16=(Ala, Ser, Tyr orTrp); Xaa at res. 18=(Glu, Lys, Gln, Met, Pro, Leu, Arg, His or Lys);Xaa at res. 19=(Gly, Glu, Asp, Lys, Ser, Gln, Arg or Phe); Xaa at res.20=(Tyr or Phe); Xaa at res. 21=(Ala, Ser, Gly, Met, Gln, His, Glu, Asp,Leu, Asn, Lys or Thr); Xaa at res. 22=(Ala or Pro); Xaa at res. 23=(Tyr,Phe, Asn, Ala or Arg); Xaa at res. 24=(Tyr, His, Glu, Phe or Arg); Xaaat res. 26=(Glu, Asp, Ala, Ser, Tyr, His, Lys, Arg, Gln or Gly); Xaa atres. 28=(Glu, Asp, Leu, Val, Lys, Gly, Thr, Ala or Gln); Xaa at res.30=(Ala, Ser, Ile, Asn, Pro, Glu, Asp, Pile, Gln or Leu); Xaa at res.31=(Phe, Tyr, Leu, Asn, Gly or Arg); Xaa at res. 32=(Pro, Ser, Ala orVal); Xaa at res. 33=(Leu, Met, Glu, Phe or Val); Xaa at res. 34=(Asn,Asp, Thr, Gly, Ala, Arg, Leu or Pro); Xaa at res. 35=(Ser, Ala, Glu,Asp, Thr, Leu, Lys, Gln or His); Xaa at res. 36=(Tyr, leis, Cys, Ile,Arg, Asp, Asn, Lys, Ser, Glu or Gly); Xaa at res. 37=(Met, Leu, Phe,Val, Gly or Tyr); Xaa at res. 38=(Asn, Glu, Thr, Pro, Lys, His, Gly,Met, Val or Arg); Xaa at res. 39=(Ala, Ser, Gly, Pro or Phe); Xaa atres. 40=(Thr, Ser, Leu, Pro, His or Met); Xaa at res. 41=(Asn, Lys, Val,Thr or Gln); Xaa at res. 42=(His, Tyr or Lys); Xaa at res. 43=(Ala, Thr,Leu or Tyr); Xaa at res. 44=(Ile, Thr, Val, Phe, Tyr, Met or Pro); Xaaat res. 45=(Val, Leu, Met, Ile or His); Xaa at res. 46=(Gln, Arg orThr); Xaa at res. 47=(Thr, Ser, Ala, Asn or His); Xaa at res. 48=(Leu,Asn or Ile); Xaa at res. 49=(Val, Met, Leu, Pro or Ile); Xaa at res.50=(His, Asn, Arg, Lys, Tyr or Gln); Xaa at res. 51=(Phe, Leu, Ser, Asn,Met, Ala, Arg, Glu, Gly or Gln); Xaa at res. 52=(Ile, Met, Leu, Val,Lys, Gln, Ala or Tyr); Xaa at res. 53=(Asn, Phe, Lys, Glu, Asp, Ala,Gln, Gly, Leu or Val); Xaa at res. 54=(Pro, Asn, Ser, Val or Asp); Xaaat res. 55=(Glu, Asp, Asn, Lys, Arg, Ser, Gly, Thr, Gln, Pro or His);Xaa at res. 56=(Thr, His, Tyr, Ala, Ile, Lys, Asp, Ser, Gly or Arg); Xaaat res. 57=(Val, Ile, Thr, Ala, Leu or Ser); Xaa at res. 58=(Pro, Gly,Ser, Asp or Ala); Xaa at res. 59=(Lys, Leu, Pro, Ala, Ser, Glu, Arg orGly); Xaa at res. 60=(Pro, Ala, Val, Thr or Ser); Xaa at res. 61=(Cys,Val or Ser); Xaa at res. 63=(Ala, Val or Thr); Xaa at res. 65=(Thr, Ala,Glu, Val, Gly, Asp or Tyr); Xaa at res. 66=(Gln, Lys, Glu, Arg or Val);Xaa at res. 67=(Leu, Met, Thr or Tyr); Xaa at res. 68=(Asn, Ser, Gly,Thr, Asp, Glu, Lys or Val); Xaa at res. 69=(Ala, Pro, Gly or Ser); Xaaat res. 70=(Ile, Thr, Leu or Val); Xaa at res. 71=(Ser, Pro, Ala, Thr,Asn or Gly); Xaa at res. 2=(Val, Ile, Leu or Met); Xaa at res. 74=(Tyr,Phe, Arg, Thr, Tyr or Met); Xaa at res. 75=(Phe, Tyr, His, Leu, Ile,Lys, Gln or Val); Xaa at res. 76=(Asp, Leu, Asn or Glu); Xaa at res.77=(Asp, Ser, Arg, Asn, Glu, Ala, Lys, Gly or Pro); Xaa at res. 78=(Ser,Asn, Asp, Tyr, Ala, Gly, Gln, Met, Glu, Asn or Lys); Xaa at res.79=(Ser, Asn, Glu, Asp, Val, Lys, Gly, Gln or Arg); Xaa at res. 80=(Asn,Lys, Thr, Pro, Val, Ile, Arg, Ser or Gln); Xaa at res. 81=(Val, Ile, Thror Ala); Xaa at res. 82=(11e, Asn, Val, Leu, Tyr, Asp or Ala); Xaa atres. 83=(Leu, Tyr, Lys or Ile); Xaa at res. 84=(Lys, Arg, Asn, Tyr, Phe,Thr, Glu or Gly); Xaa at res. 85=(Lys, Arg, His, Gln, Asn, Glu or Val);Xaa at res. 86=(Tyr, His, Glu or Ile); Xaa at res. 87=(Arg, Glu, Gln,Pro or Lys); Xaa at res. 88=(Asn, Asp, Ala, Glu, Gly or Lys); Xaa atres. 89=(Met or Ala); Xaa at res. 90=(Val, Ile, Ala, Thr, Ser or Lys);Xaa at res. 91=(Val or Ala); Xaa at res. 92=(Arg, Lys, Gln, Asp, Glu,Val, Ala, Ser or Thr); Xaa at res. 93=(Ala, Ser, Glu, Gly, Arg or Thr);Xaa at res. 95=(Gly, Ala or Thr); Xaa at res. 97=(His, Arg, Gly, Leu orSer). Further, after res. 53 in rBMP3b and mGDF-10 there is an Ile;after res. 54 in GDF-1 there is a T; after res. 54 in BMP3 there is a V;after res. 78 in BMP-8 and Dorsalin there is a G; after res. 37 inhGDF-1 there is Pro, Gly, Gly, Pro.

Generic Sequence 10 (SEQ ID NO: 7) includes all of Generic Sequence 9(SEQ ID NO: 6) and in addition includes the following sequence (SEQ IDNO: 9) at its N-terminus:

SEQ ID NO: 9 Cys Xaa Xaa Xaa Xaa   1               5Accordingly, beginning with residue 6, each “Xaa” in Generic Sequence 10is a specified amino acid defined as for Generic Sequence 9, with thedistinction that each residue number described for Generic Sequence 9 isshifted by five in Generic Sequence 10. Thus, “Xaa at res. 1=(Tyr, Phe,His, Arg, Thr, Lys, Gln, Val or Glu)” in Generic Sequence 9 refers toXaa at res. 6 in Generic Sequence 10. In Generic Sequence 10, Xaa atres. 2=(Lys, Arg, Gln, Ser, His, Glu, Ala, or Cys); Xaa at res. 3=(Lys,Arg, Met, Lys, Thr, Leu, Tyr, or Ala); Xaa at res. 4=(His, Gln, Arg,Lys, Thr, Leu, Val, Pro, or Tyr); and Xaa at res. 5=(Gln, Thr, His, Arg,Pro, Ser, Ala, Gln, Asn, Tyr, Lys, Asp, or Leu).

As noted above, certain currently preferred bone morphogenic polypeptidesequences useful in this invention have greater than 60% identity,preferably greater than 65% identity, with the amino acid sequencedefining the preferred reference sequence of hOP-1. These particularlypreferred sequences include allelic and phylogenetic counterpartvariants of the OP-1 and OP-2 proteins, including the Drosophila 60Aprotein. Accordingly, in certain particularly preferred embodiments,useful morphogenic proteins include active proteins comprising pairs ofpolypeptide chains within the generic amino acid sequence hereinreferred to as “OPX” (SEQ ID NO: 3), which defines the seven cysteineskeleton and accommodates the homologies between several identifiedvariants of OP-1 and OP-2. As described therein, each Xaa at a givenposition independently is selected from the residues occurring at thecorresponding position in the C-terminal sequence of mouse or human OP-1or OP-2.

Cys Xaa Xaa His Glu Leu Tyr Val Ser Phe Xaa Asp Leu Gly Trp Xaa Asp Trp 1              5                   10                  15Xaa Ile Ala Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly Glu Cys Xaa Phe Pro    20                  25                  30                  35Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala Ile Xaa Gln Xaa Leu Val His Xaa        40                  45                  50                  55Xaa Xaa Pro Xaa Xaa Val Pro Lys Xaa Cys Cys Ala Pro Thr Xaa Leu Xaa Ala            60                  65                  70Xaa Ser Val Leu Tyr Xaa Asp Xaa Ser Xaa Asn Val Ile Leu Xaa Lys Xaa Arg75                  80                  85                  90Asn Met Val Val Xaa Ala Cyn Gly Cys His         95                  100wherein Xaa at res. 2=(Lys or Arg); Xaa at res. 3=(Lys or Arg); Xaa atres. 11=(Arg or Gln); Xaa at res. 16=(Gln or Leu); Xaa at res. 19=(Ileor Val); Xaa at res. 23=(Glu or Gln); Xaa at res. 26=(Ala or Ser); Xaaat res. 35=(Ala or Ser); Xaa at res. 39=(Asn or Asp); Xaa at res.41=(Tyr or Cys); Xaa at res. 50=(Val or Leu); Xaa at res. 52=(Ser orThr); Xaa at res. 56=(Phe or Leu); Xaa at res. 57=(Ile or Met); Xaa atres. 58=(Asn or Lys); Xaa at res. 60=(Glu, Asp or Asn); Xaa at res.61=(Thr, Ala or Val); Xaa at res. 65=(Pro or Ala); Xaa at res. 71=(Glnor Lys); Xaa at res. 73=(Asn or Ser); Xaa at res. 75=(Ile or Thr); Xaaat res. 80=(Phe or Tyr); Xaa at res. 82=(Asp or Ser); Xaa at res.84=(Ser or Asn); Xaa at res. 89=(Lys or Arg); Xaa at res. 91=(Tyr orHis); and Xaa at res. 97=(Arg or Lys).

In still another preferred embodiment, useful osteogenically activeproteins have polypeptide chains with amino acid sequences comprising asequence encoded by a nucleic acid that hybridizes, under low, medium orhigh stringency hybridization conditions, to DNA or RNA encodingreference morphogen sequences, e.g., C-terminal sequences defining theconserved seven cysteine domains of OP-1, OP-2, BMP2, 4, 5, 6, 60A,GDF3, GDF6, GDF7 and the like. As used herein, high stringenthybridization conditions are defined as hybridization according to knowntechniques in 40% formamide, 5×SSPE, 5×Denhardt's Solution, and 0.1% SDSat 37° C. overnight, and washing in 0.1×SSPE, 0.1% SDS at 50° C.Standard stringence conditions are well characterized in commerciallyavailable, standard molecular cloning texts. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984): Nucleic Acid Hybridization (B. D. Hames & S. J.Higgins eds. 1984); and B. Perbal, A Practical Guide To MolecularCloning (1984).

As noted above, proteins useful in the present invention generally aredimeric proteins comprising a folded pair of the above polypeptides.Such morphogenic proteins are inactive when reduced, but are active asoxidized homodimers and when oxidized in combination with others of thisinvention to produce heterodimers. Thus, members of a folded pair ofmorphogenic polypeptides in a morphogenically active protein can beselected independently from any of the specific polypeptides mentionedabove.

The bone morphogenic proteins useful in the materials and methods ofthis invention include proteins comprising any of the polypeptide chainsdescribed above, whether isolated from naturally-occurring sources, orproduced by recombinant DNA or other synthetic techniques, and includesallelic and phylogenetic counterpart variants of these proteins, as wellas muteins thereof, and various truncated and fusion constructs.Deletion or addition mutants also are envisioned to be active, includingthose which may alter the conserved C-terminal six or seven cysteinedomain, provided that the alteration does not functionally disrupt therelationship of these cysteines in the folded structure. Accordingly,such active forms are considered the equivalent of the specificallydescribed constructs disclosed herein. The proteins may include formshaving varying glycosylation patterns, varying N-termini, a family ofrelated proteins having regions of amino acid sequence homology, andactive truncated or mutated forms of native or biosynthetic proteins,produced by expression of recombinant DNA in host cells.

The bone morphogenic proteins contemplated herein can be expressed fromintact or truncated cDNA or from synthetic DNAs in prokaryotic oreukaryotic host cells, and purified, cleaved, refolded, and dimerized toform morphogenically active compositions. Currently preferred host cellsinclude, without limitation, prokaryotes including E. coli, oreukaryotes including yeast, or mammalian cells, such as CHO, COS or BSCcells. One of ordinary skill in the art will appreciate that other hostcells can be used to advantage. Detailed descriptions of the bonemorphogenic proteins useful in the practice of this invention, includinghow to make, use and test them for osteogenic activity, are disclosed innumerous publications, including U.S. Pat. Nos. 5,266,683 and 5,011,691,the disclosures of which are incorporated by reference herein, as wellas in any of the publications recited herein, the disclosures of whichare incorporated herein by reference.

Thus, in view of this disclosure and the knowledge available in the art,skilled genetic engineers can isolate genes from cDNA or genomiclibraries of various different biological species, which encodeappropriate amino acid sequences, or construct DNAs fromoligonucleotides, and then can express them in various types of hostcells, including both prokaryotes and eukaryotes, to produce largequantities of active proteins capable of stimulating endochondral bonemorphogenesis in a mammal.

II. Binding Agent Considerations

As already explained, “binding agent”, as used herein, means anyphysiologically-compatible material which, when admixed with osteogenicprotein and matrix as defined herein promotes bone and/or cartilageformation. In certain currently preferred embodiments, binding agentspromote such repair using less osteogenic protein than standardosteogenic devices. Among the other characteristics of a preferredbinding agent is an ability to render the device: pliable, shapeableand/or malleable; injectable; adherent to bone, cartilage, muscle andother tissues; resistant to disintegration upon washing and/orirrigating during surgery; and, resistant to dislodging during surgery,suturing and post-operatively, to name but a few. Additionally, in acurrently preferred embodiment, binding agent can achieve theaforementioned features and benefits when present in relatively lowproportions. For example, a currently preferred improved devicecomprises approximately 1 part binding agent and approximately 5 partsmatrix. Another currently preferred device comprises 1 part bindingagent and 3 parts matrix. As exemplified herein, improved devices ofwidely divergent proportions can induce bone and cartilage formation.Exemplified herein are improved devices having parts of binding agent toparts of matrix ranging from approximately 1:1 to 4:1 up to andincluding at least 10:1, as well as from approximately 1:2 to 1:5, up toand including at least 1:10, and further including 1:25 to 1:50. Anyproportion of binding agent to matrix can be used to practice theinstant invention. All that is required is admixing binding agent withmatrix and osteogenic protein so as to achieve bone and cartilageformation. As discussed below, certain binding agents can be used inequal or greater proportions relative to matrix, but such agents shouldbe tested as taught herein to measure any matrix dilution effects.

Those binding agents contemplated as useful herein include, but are notlimited to: art-recognized gelling agents, suspending agents,viscosity-producing agents and emulsifying agents. In particular,art-recognized agents, such as cellulose gum derivatives and sodiumalginate, gelatin powder and dextrans can be used. More particularly,cellulosic agents such as alkylcelluloses, including agents such asmethylcellulose, methylhydroxyethylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose, and hydroxyalkylcelluloses, to name but a few.Currently among the most preferred is carboxymethylcellulose, includingthe sodium salt thereof. As exemplified below, other binding agentssuitable for use in the instant invention include, but are not limitedto, dextran, mannitol, white petrolatum, sesame oil and admixturesthereof.

Finally, also among the most preferred binding agents is a fibrin glue,which comprises a mixture of mammalian fibrinogen and thrombin. Asexemplified herein, fibrin glue as a preferred binding agent cancomprise wide ranges of fibrinogen and thrombin. In certain embodimentscomprising 1 part fibrin glue and 25 parts β-TCP matrix, thrombincontent can range from about 2.0 U to 25 U, preferably 5 U to 10 U andmost preferably about 2.5 U to 5 U. In certain other devices comprisingfibrin glue and collagen matrix, thrombin content can range from about2.0 U to 25 U; preferably 5 U to 25 U, more preferably 2.0 U to 10 U andmost preferably 2.5 U to 5 U. Fibrinogen content can range from about 40mg per 1000 mg β-TCP, for example. In a collagen-containing improveddevice, fibrinogen content can range from about 20 mg per 1000 mgcollagen to about 180 mg per 1000 mg collagen, for example.

In view of the teachings set forth herein, the artisan can identifysuitable equivalents of the above-identified binding agents using merelyroutine experimentation and ordinary skill. Suitable binding agentcandidates can be identified, characterized, tested and then used inosteogenic devices as set forth below.

In general, agents which are recognized in the art as suspending orviscosity-producing agents in pharmaceutical technologies are suitablefox use as a binding agent in the instant invention. Reference manualssuch as the USP XXII-NF XVII (The Nineteen Ninety U.S. Pharmacopeia andthe National Formulary (1990)) categorize and describe such agents. Forexample, binding agent candidates are those described as useful asemulsifying agents, gel-forming agents, binders, or viscosity-producingagents for injectables and parenterals. Other candidates are agents usedto suspend ingredients for topical, oral or parenteral administration.Yet other candidates are agents useful as tablet binders, disintegrantsor emulsion stabilizers. Still other candidates are agents used incosmetics, toiletries and food products. When used for any of theforegoing applications, candidate agents are described as typicallypresent for conventional applications at concentrations ranging fromapproximately 0.1 to 6.0%. At the highest standard concentrations(4-6%), certain of the foregoing candidate agents are used in thepharmaceutical industry, for example, to produce medicaments in the formof gels or pastes.

Thus the skilled artisan can identify binding agent candidatesaccordingly and can similarly recognize equivalents of the preferredbinding agents specifically identified herein using only routine skilland routine experimentation. Having identified a suitable candidate(s),the skilled artisan can then follow the guidelines set forth below as tofinal selection of a preferred binding agent.

Based on studies similar to those described herein, examples of suitablebinding agents useful in the improved devices disclosed herein include,but are not limited to: mannitol/dextran combination; dextran alone;mannitol/white petrolatum combination; and sesame oil. Amannitol/dextran-containing improved device was formulated as follows.One part dextran 40, 3 parts mannitol, 1 part OP device. Such improveddevices were formulated with 2.5 mg osteogenic protein per g collagen orper 0.5 g collagen, thereby varying the dose of osteogenic protein. Foruse in the instant method, the formulation was wetted with approximately0.8 ml saline per 2.5 g mannitol/dextran-containing device. Next, adextran alone-containing device was formulated from either 4 partsdextran or 1 part dextran to 1 part OP device, and wetted withapproximately 0.8 ml saline per 2.0 g device. Dextran can range from3,000 to 40,000 m.w. Next, a mannitol/white petrolatum device wasformulated from 1.5 parts mannitol, 1.5 parts petrolatum, and 1 part OPdevice. This formulation does not require wetting. Finally, a sesameoil-containing improved device was formulated from 1 part oil and 1 partOP device. This formulation does not require wetting. Theabove-described improved devices illustrate the range of: specificbinding agents, proportions in improved devices, and volumes of wettingagent which can be used in the improved devices of the instantinvention. Chemistries, proportions and wetting requirements are varied,yet all are within the skill of the art. Each of the aforementionedimproved devices induced bone formation (as measured by calcium contentand % bone) when tested in the rat subcutaneous bioassay describedherein.

A. CMC as a Binding Agent

As taught herein, carboxymethylcellulose (CMC) is a currently preferredbinding agent. CMC is commercially available from suppliers such as, butnot limited to: Hercules Inc., Aqualon®Division, Delaware; FMCCorporation, Pennsylvania; British Celanese, Ltd., United Kingdom; andHenkel KGaA, United Kingdom. Carboxymethylcellulose sodium is the sodiumsalt of a polycarboxymethyl ether of cellulose with a typical molecularweight ranging from 90,000-700,000. CMC was identified as a candidatebinding agent based, in part, on the following: CMC is widely used inoral and topical pharmaceutical formulations as a viscosity-increasingagent. CMC is also used in cosmetics, toiletries and foods as anemulsifying agent (0.25-1.0%); gel forming agent (4.0-6.0%), injectable(0.05-0.75%), and tablet binder (1.0-6.0%).

While the foregoing characteristics are suggestive of suitability as abinding agent, the experiments detailed below confirmed that CMC wassuitable for use in the improved osteogenic devices disclosed herein.Such confirmatory experiments were necessary because none of theaforementioned applications are similar to the repair of bone orcartilage for which the improved osteogenic devices disclosed herein areuseful. For example, none of the aforementioned applications require CMCabove 6%, yet a currently preferred implantable improved device of theinstant invention comprises more than approximately 6% (w/w) CMC andpreferably at least approximately 10%, more preferably approximately12-20%, with approximately about 16% (w/w) or 1 part CMC to 5 partsstandard osteogenic device being among the most currently preferred foran implantable device. These approximate percentages are based oncalculations of total weight of matrix admixed with binding agent,excluding osteogenic protein and wetting agent.

Of significance to practice of the instant invention is the fact thatvarious grades of carboxymethylcellulose sodium are commerciallyavailable which have differing viscosities. Viscosities of variousgrades of carboxymethylcellulose sodium are reported and shown in Table1 below (see, Handbook of Pharmaceutical Excipients (2nd Edition),American Pharmaceutical Association & Royal Pharmaceutical Society ofGreat Britain).

TABLE 1 Standard Viscosity Grades of CarboxymethylcelluloseConcentration Viscosity Grade (% w/v) (cP) Low viscosity 4  50-200Medium viscosity 2 400-800 High viscosity 1 1500-3000

A number of grades of carboxymethylcellulose are commercially available,the most frequently used grade having a degree of substitution (DS) of0.7. The DS is defined as the average number of hydroxyl groupssubstituted per anhydroglucose unit. It is this DS which determines theaqueous solubility of the polymer. The degree of substitution and thestandard viscosity of an aqueous solution of stated concentration isindicated on any carboxymethylcellulose sodium labelling. Low viscosityCMC (Aqualon® Division, Hercules Inc., Wilmington, Del.) is currentlypreferred. The currently preferred degrees of substitution range from0.65-0.90 (DS=0.7, Aqualon® Type 7L).

As described above, CMC is available in several grades—low, medium andhigh viscosity. In this regard, the viscosity of thecarboxymethylcellulose (CMC) used to formulate an improved osteogenicdevice was determined to be critical for bone formation. Contrary toteachings in the art, it has now been discovered that high viscosity CMCadversely affects bone formation when used in an improved osteogenicdevice comprising a matrix as defined herein. U.S. Pat. No. 5,587,897(“the '897 patent”) teaches the use of high viscosity (2480 cP) (seeTable 1 above) CMC to induce bone formation. The devices in the '897patent, however, require a synthetic polymer matrix, rather than abiological matrix such as collagen. Unexpectedly, when a biologicalmaterial such as collagen is used as a matrix, the improved device mustbe formulated with low viscosity CMC (approximately 10-50 cP, or 50-200)in order to induce bone and/or cartilage formation, as taught herein.

Toxicity Study Using a CMC Device

A toxicity study was conducted comparing a CMC-containing improveddevice to that of a standard device. The standard device was preparedwith 2.5 mg OP-1/gram collagen matrix. The CMC containing improveddevice was prepared by adding low viscosity CMC (Aqualon®) to a standarddevice at the ratio of 1:5 followed by irradiation. 25 mg aliquots of astandard device or mock device (i.e., no osteogenic protein) and 30 mgaliquots of CMC containing improved device or mock CMC device wereimplanted at a rat sub-cutaneous site as described elsewhere herein (oneimplant per animal). Three implants from each formulation were removedat 7 days, 14 days, 21 days and 28 days post-implantation, and evaluatedhistologically for bone and cartilage formation and for local tissuereaction. No adverse cellular reaction was observed, and there was noevidence to indicate any adverse effects of CMC, as determined byevaluating inflammation and fibrous formation. The histological profileof the CMC-containing-improved device was generally similar to thestandard OP-device. Serum calcium and alkaline phosphatase levels,measured using standard teachings also followed that of the standardosteogenic device. Finally, using standard toxicity analyses on immatureand mature rats, no significant lesions were detected.

Improved Device Bioactivity Studies

Based on a series of routine studies described below, the bioactivity ofa standard osteogenic device was not adversely affected by admixturewith CMC. Rather, bioactivity is at least comparable for both deviceconfigurations, but the ability to manipulate the deviceintraoperatively and to retain the device at the defect site duringsurgery and wound closure is enhanced by CMC. For these studies,irradiated CMC was added to the standard device prior to implantation.

Briefly, two studies were conducted measuring the in vivo release ofOP-1 from a standard device+/−CMC. In one experiment, 75 mg ofirradiated device+/−15 mg of irradiated CMC were implanted in asubcutaneous site in rats as described herein. The implanted deviceswere removed 1 hour, 1 day, 3 days and 6 days after implantation,followed by extraction with 8M urea buffer; the OP-1 content wasanalyzed by routine ELISA and western blot analysis. OP-1 device (notimplanted into the animals) was extracted with 8M urea buffer and usedas the internal standard. In general, the kinetics of in vivo release ofOP-1 from the standard OP device and the CMC-containing improved devicewere similar. The observation that there is no difference in OP-1sequestration or retention by a standard device versus an improveddevice containing a combination of collagen matrix and CMC is anunexpected result. It has been reported that, when combined withnon-biological polymeric matrices, CMC acts to sequester osteogenicprotein. (See, for example, U.S. Pat. No. 5,597,897).

In vitro studies were also conducted. In these studies, released OP-1was measured in contrast to the above-described in vivo studies, inwhich OP-1 remaining in the device was measured. In one study, 25 mg ofOP device or CMC device was wetted with saline. 1 ml of bovine serum wasthen added to each device, and the devices were incubated at 37° C. Thesupernatant was removed and replaced with fresh serum at 1 and 3 hours.At 6 hours 8M urea was added to extract any OP-1 still associated withthe device. OP-1 concentrations in the supernatants were analyzed byroutine ELISA and western blot techniques. Both the standard OP deviceand the CMC-containing improved device had similar protein releasekinetics for the six hours studied. Again, these results were unexpectedin view of earlier reports that CMC acts to sequester osteogenic proteinand thereby retard and/or prevent its release from admixtures withsynthetic, polymeric matrices. (See, for example, U.S. Pat. No.5,597,897).

In conclusion, CMC does not substantially inhibit the retention orrelease of OP-1 from a collagen matrix-containing osteogenic device invivo or in vitro.

Stability Studies

A study (see Table 2) was conducted comparing the stability of thestandard OP device to a standard device containing CMC. Based on both invitro analyses and a bone-forming bioassay (described elsewhere herein),the CMC-containing improved device was observed to be at least as stableas the standard device when stored at 30 degrees for one year. The dataalso suggest that CMC can be premixed with the standard OP device andterminally sterilized for a unitary product configuration. Such aunitary product is useful for repair of local bone and cartilage defectsas exemplified below.

TABLE 2 Stability Of Various Osteogenic Device Formulations OP-1 OP-1OP-1 OP-1 Pre-Irradiation Post-Irradiation Recovery Recovery RecoveryRecovery OP-1 OP-1 after 4 after 3 after 6 after 12 FormulationRecovery* Recovery weeks months months months Standard 91% 63% 66% 58%47.4% 37.4% Device CMC Device 77% 57% 53% 50% 43.5% 34.8% *Based ontheoretical OP-1 content of 2.5 mg/gram

During formulation of a standard device containing CMC, the CMC andosteogenic proteins may be sterilized separately, for example, byexposure to gamma irradiation and then the sterilized componentscombined to produce the standard device containing CMC. Furthermore, theCMC can be premixed with the standard OP device and the resultingformulation sterilized, for example, by exposure to gamma irradiation.The latter process is referred to in the art as terminal sterilizationand has been used to sterilize other osteogenic devices. See, forexample, PCT/US96/10377, published as WO 96/40297 on Dec. 19, 1996, andU.S. Pat. No. 5,674,292 issued on Oct. 7, 1997 the disclosures of whichare incorporated by reference. As used herein, the terms “sterilization”and “sterilized” refer to a process using either physical or chemicalmeans for eliminating substantially all viable organisms, especiallymicro-organisms, viruses and other pathogens, associated with the deviceof the invention. The sterilized devices of the invention preferablyhave a sterility assurance level of 10⁻⁶ as determined by Federal DrugAdministration (FDA) standards. In the case of gamma irradiated devices,for example, the appropriate dosages of irradiation necessary forsterilizing a particular device can be determined readily by consultingthe reference text “Associate for the Advancement of MedicalInstrumentation Guidelines,” published 1992. Guidelines are providedtherein for determining the radiation dose necessary to achieve a givensterility assurance level for a particular bioburden of the device.Dosages for sterilizing devices of the invention preferably are withinthe range of about 0.5 to about 4.0 megarods and most preferably arewithin the range of about 2.0 to about 3.5 megarods.

Additionally, a study was conducted to evaluate the short term stabilityof an osteogenic device to which CMC and saline had been added. Thestudy used a standard device to which 200 mg of separately packaged,irradiated CMC was added. Samples of CMC-containing improved device wereremoved and wet with saline. At 0, 1, 3, 6 and 22 hours the OP-1 wasextracted with 8 M urea buffer and analyzed by reverse-phase HPLC underreducing conditions. The extracts were also analyzed for OP-1 biologicalactivity a standard cell-based assay for measuring alkaline phosphatase.The data indicated that OP-1 retains biological activity under theseconditions. These data also suggested that the configuration ofCMC-containing improved device resulting from admixture of thesecomponent parts (standard osteogenic device/CMC/saline) is useable forseveral hours after it has been prepared, providing the practitionerwith significant intraoperative time during which the product remainsefficacious.

Testing of Binding Agent Integrity and Other Characteristics

Art-recognized USP methods were used for identification andcharacterization of bulk binding agents such as CMC. Tests includedtests for chemical identity, viscosity, pH, loss on drying and heavymetals. Material was also tested for bioburden prior to sterilization,as well as endotoxins, pH, appearance and sterility after irradiation. Astability study was conducted to monitor the viscosity, appearance andpH of the irradiated material. All levels and characteristics wereacceptable as determined using standard methods and techniques.

For example, CMC (Aqualon®-low viscosity) was evaluated for bioburdenand endotoxin content. Aqualon® CMC, Lot FP10 12342, was evaluated forthe presence of endotoxins (LAL) using the Kinetic Chromogenic LAL assayfrom BioWhittaker (Walkersville, Md., 21793).

“Bioburden” can be measured as follows. For example 200 mg samples ofCMC were solubilized in 100 ml of phosphate buffered water and filteredthrough 0.45 μm filters. The filters were placed on a TSA plate andincubated for 48 hours. Two samples of solubilized CMC were inoculatedwith 10-100 CPUs of Bacillus subtilis to be used as growth controls. Thedata suggest that the bioburden of the CMC is low, and that CMC does notinterfere in the analysis by killing bacteria or inhibiting cell growth.

CMC Characterization Post-Irradiation

A study was conducted comparing the viscosity of CMC pre- andpost-irradiation (gamma irradiation, 2.5-3.0 mega rads). The dataindicated that, as reported in the art, viscosity decreases afterirradiation. While this does not affect bioactivity or its overallutility as a binding agent (see studies set forth herein), the skilledpractitioner should take this feature into consideration when assessingviscosity or fluidity properties of an improved osteogenic device. Astudy was also conducted to evaluate the stability of irradiated CMC.The results indicated that irradiated CMC was stable for at least sixmonths at both 4 and 30° C. Viscosity was measured as the parameter ofstability. Similar analyses and assessments can be carried out for otherbinding agents or device materials used in a desired formulation.

B. Fibrin Glue as a Binding Agent

As taught herein, “fibrin glue” is another currently preferred bindingagent. Fibrin glue comprises a mixture of mammalian fibrinogen andthrombin. Human fibrinogen is commercially available in products suchas, but not limited to: Tissucol® (ImmunoAG, Vienna, Austria),Beriplast® (Behringwerke, Marburg, Germany), Biocoll® (Centre deTransfusion Sanguine de Lille (Pours, France) and Transglutine® (CNTSFractionation Centre, Strasbourg, France). Human thrombin iscommercially available from ImmunoAG, Vienna, Austria. Fibrin glue mayalso be made of fibrinogen and thrombin from other mammalian sources,such as, for example, bovine and murine sources.

Fibrin glue was identified as a candidate binding agent based on itsgel-like properties and its improved handling characteristics whenadmixed with a matrix material, such as, for example, collagen or β-TCP.Fibrin glue was also shown to elicit a low inflammatory response (seebelow) and to promote bone formation.

Toxicity Study Using a Fibrin Glue Device

A toxicity study was conducted comparing a fibrin glue-containingimproved device to that of a standard device. The standard device wasprepared by mixing 10 μg OP-1 in 47.5% ethanol/0.01% TFA and 25 mgcollagen and lyophilizing the mixture overnight. The standard device waswetted with 100 μL phosphate buffered saline (PBS) prior toimplantation. The fibrin glue-containing improved device was prepared byadding 50 μL bovine fibrinogen (Sigma F8630, 10 mg/ml) and 50 μL bovinethrombin (50 U/mL) to the standard device, prepared as described above,immediately prior to implantation. The standard device and the fibringlue-containing improved device were then implanted at a ratsub-cutaneous site as described elsewhere herein. The implants wereevaluated histologically for bone and cartilage formation and for localtissue reaction. The fibrin glue-containing improved device elicited alow inflammatory response and low fibrous formation. The histologicalprofile of the fibrin glue-containing improved device was generallysimilar to that of the standard device. There did not appear to be anycorrelation between the inflammatory response and the ability of thefibrin glue-containing improved device to promote bone formation.

Improved Device Bioactivity Studies

A study was done to evaluate the release kinetics of OP-1 from a fibringlue-containing improved device at different thrombin concentrations invitro. Release kinetics were improved by the addition of larger amountsof thrombin. For this study, 12.5 μl, of OP-1 in a 5% lactose solutionwas mixed with 50 mg β-TCP, 25 μL human fibrinogen, and either 25 U/mLof human thrombin or 50 U/mL human thrombin. The mixtures weretransferred to a glass vial and 1 mL of calf serum added to each. Thesamples were then allowed to incubate at 37° C./60 rpm. Serum sampleswere taken and analyzed by routine ELISA at 0-1 hours, 1-3 hours, 3-5hours and 5-24 hours. The results are summarized in the table below.

TABLE 2A % OP-1 % OP-1 % OP-1 % OP-1 Thrombin release at release atrelease at release at Total % OP-1 conc. 0-1 hours 1-3 hours 3-5 hours5-24 hours release 25 U/mL 6.4 +/− 0.7 5.2 +/− 0.4 3.0 +/− 0.6 13.0 +/−0.6 27.5 +/− 2.2 50 U/ml 9.9 +/− 2.1 5.8 +/− 0.7 3.3 +/− 0.5 15.1 +/−1.7 34.1 +/− 4.8

III. Formulation and Delivery Considerations

General Considerations

The devices of the invention can be formulated using routine methods.All that is required is determination of the desired final concentrationof osteogenic protein per device, keeping in mind that the deliveredvolume of device can be, but is not necessarily required to be, lessthan the volume at the defect site. The desired final concentration ofprotein will depend on the specific activity of the protein as well asthe type, volume, and/or anatomical location of the defect.Additionally, the desired final concentration of protein can depend onthe age, sex and/or overall health of the recipient. Typically, for acritical size segmental defect approximately at least 2.5 cm in length,0.5-1.75 mg osteogenic protein has been observed using the standarddevice to induce bone formation sufficient to repair the gap. In thecase of a non-critical size defect or a fresh fracture, approximately0.1-0.5 mg protein has been observed using the standard osteogenicdevice to repair the defect. In general, protein concentrations for usewith preferred matrices described herein can range from about 0.4 mg toabout 3.0 mg per device. Optimization of dosages requires no more thanroutine experimentation and is within the skill level of one of ordinaryskill in the art.

As exemplified herein, osteogenic protein and a binding agent such ascarboxymethylcellulose (low viscosity, Aqualon®) or fibrin glue can beadmixed to form a putty. In some embodiments, saline is added to bindingagent to form a paste or putty in which an osteogenic protein such asOP-1 is dispersed. A paste configuration can be used to paint thesurfaces of a defect, such as a cavity. Pastes can be used to paintfracture defects, chondral or osteochondral defects, as well as bonedefects at a prosthetic implant site. A more fluid configuration can beinjected or extruded into or along the surfaces of a defect, in a mannersimilar to extruding toothpaste or caulking from a tube, such that abead of device is delivered along the length of the defect site.Typically, the diameter of the extruded bead is determined by the typeof defect as well as the volume of the void at the defect site.

As mentioned above, other binding agents as defined herein can be usedto formulate a device with a configuration like putty. As will beobvious to the skilled artisan, such a configuration results fromadjusting the proportion of carrier to wetting agent, with less wettingagent producing a drier device and more producing a wetter device. Theprecise device configuration suitable to repair a defect will at leastdepend on the type of defect and the size of the defect. The skilledartisan will appreciate the variables.

A. CMC as a Binding Agent—Formulation Studies

Based on the following type of studies, it was established thatapproximately 0.2 g of CMC to approximately 1.0 g standard osteogenicdevice yields an improved device with the currently preferred handlingproperties. Varying ratios of CMC and collagen were combined and thenwet with saline. Each resulting mixture of CMC and matrix was suspendedin a 15 ml conical centrifuge tube of water and placed on a rotaryshaker (100 rpm). Settling time was recorded when loosened or releasedcollagen matrix particles settled to a predetermined mark on the tube.The data summarized in Table 3 and FIG. 1 suggest that a range ofapproximately 0.15 to 0.25 g CMC/g collagen can maximize cohesiveness,integrity and handling properties.

TABLE 3 Effect Of CMC/Collagen Ratio On Dispersion Time g CMC/g SettlingCollagen Time 0.20 g  19 min 0.19  17 0.18   6 0.15   4 0.12 0.5 sec

The preferred amount of saline for wetting the CMC device was alsostudied. In this study, approximately 0.2 g of CMC were mixed withapproximately 1 g standard osteogenic device. Varying amounts of salinewere added, and the consistency of the resulting device was noted. Thequalitative and quantitative results from this study are summarized inTable 4 and FIG. 2, respectively. Generally, these data illustrate thatthere is a range of wetting agent volumes which can accommodate thepractitioner while enabling the device to retain its integrity andcohesiveness. For a binding agent like CMC, the data suggest that morethan approximately 1.5 ml, approximately 1.8 to 2.5 ml of saline, is thecurrently preferred wetting volume (for approximately 1 gram of deviceadmixed with approximately 200 mg of a binding agent such as CMC) toachieve an implantable device with the currently preferred puttyconsistency. Amounts of saline in excess of this achieve an injectabledevice with the currently preferred fluid consistency. As exemplifiedelsewhere herein, an implantable device configuration is suitable foruse at an open defect site, while an injectable device configuration issuitable for use at a closed defect site. In terms of gram equivalents,approximately 0.5 g to approximately 3.0 g saline has been determined toyield improved devices with desirable consistencies; the higher theweight, the more injectable is the configuration.

TABLE 4 Wetting of the CMC-Containing Device 1 gram Standard Device plus200 mg Amount of CMC Saline Added Observations  1.5 ml Dry 1.75 Rollsinto a ball; paste  2.0 Currently preferred handling consistency; putty.Rolls into a ball.  2.5 Acceptable handling consistency; stillputty-like. 2.75 Leaves small particles of matrix on vessel wall.  3.0Sticky; soft paste.  3.5 Sticky; soft paste. 3.75 Loose paste.  4.0Consistency same as above. 4.25 Liquid

In certain embodiments of the present invention, preparation of theactual improved osteogenic device can occur immediately prior to itsdelivery to the defect site. As exemplified herein, CMC-containingimproved devices can be prepared on-site, suitable for admixingimmediately prior to surgery. In one embodiment, low viscosity CMC(Aqualone) was packaged and irradiated separately from the osteogenicprotein. OP-1 and collagen matrix. The OP-1 protein in collagen matrixthen was admixed with the binding agent. Devices prepared in this mannerwere observed to be at least as biologically active as the standarddevice without CMC.

B. Fibrin Glue as a Binding Agent—Formulation Studies

Based on the following type of studies, it was established thatapproximately 500 μL fibrinogen (80 mg/mL in PBS at pH 7.4) and 500 μLthrombin (50 U/mL or 25 U/mL in 0.9% NaCl) added to approximately 1 μmof β-TCP yields an improved device with the currently preferred handlingproperties. Issues related to handling properties include the clottingtime of the fibrin glue and the consistency of the fibringlue-containing improved device. A device having a consistency of amoldable putty is preferred. Once the glue clots, it becomes moredifficult to change the shape of the putty. A longer clotting time is,therefore, also a preferred feature of the device.

The clotting time of bovine fibrin glue was determined by mixing 20 μLbovine fibrinogen solution (80 mg/mL in PBS at pH 7.4) with 20 μL bovinethrombin solution (500 U/mL or 25 U/mL in saline) continuously in aweight boat using a capillary glass rod. The clotting times were alsoevaluated with or without addition of 0.6% CaCl₂ solution. The resultsare shown in the following table:

TABLE 4A Clotting time Clotting time Thrombin in seconds in seconds(U/mL) w/o CaCl₂ with 0.6% CaCl₂ 500 23 +/− 2 20 +/− 2 250 40 +/− 5 29+/− 2 100 61 +/− 2 49 +/− 1 50 90 +/− 2 74 +/− 1 25 — 185 +/− 5 

The consistency of the fibrin glue-containing device was evaluated usinga device containing β-TCP as an exemplary matrix. Differing amounts ofbovine fibrin glue were added to 100 mg or 1000 mg of β-TCP granules andthe consistency determined. The results are summarized in the tablebelow:

TABLE 4B Fibrinogen μL (80 mg/mL Amount of in PBS, pH β-TCP 7.4)Thrombin μL Consistency 100 mg 12.5 12.5 (100 U/mL in β-TCP not wettedsaline w/0.6% completely CaCl₂) 100 mg 25 25 (100 U/mL in slightlymoldable saline w/0.6% CaCl₂) 100 mg 50 50 (100 U/mL in putty, moldableat saline w/0.6% 1-2 min. CaCl₂) 100 mg 50 50 (50 U/mL in moldable puttysaline w/0.6% CaCl₂) 100 mg 50 50 (5 U/mL in saline clotting very slow,w/0.6% CaCl₂) particles separated 1000 mg  500 500 (100 U/mL in mixingtoo slow, saline w/0.6% inhomogenous, CaCl₂) particles separated 1000mg  500 500 (50 U/mL in putty, moldable at saline) 1-2 min. 1000 mg  500500 (25 U/mL in putty, moldable at saline) 2-3 min.

As can be seen by these two studies, a fibrin glue-containing device ofapproximately 500 μL fibrinogen (80 mg/mL in PBS at pH 7.4) and 500 μLthrombin (50 U/mL or 25 U/mL in 0.9% NaCl) added to approximately 1 μmof β-TCP has a clotting time and consistency suitable for the improvedosteogenic device.

TABLE 4C Representative Information Relating to the Composition ofFibrin Glue Autocolle ® Tissucol ® Beriplast ® Transglutine ® Biocoll ®Fibrinogen 50-65  70-110  20-140 >70 116 +/− 2.4   (mg/mL) Fibronectin 4-10 2-9 5.9 +/− 0.51 (mg/mL) Factor XIII 25-30 10-50 40-60  35 +/−2.88 (PEU) Thromboglobulin 250-400 (μg/mL) PDGF (ng/mL) 350 TGF (ng/mL)750 Plasminogen  40-120 31 (μg/mL) Aprotinin 3000 (KIU/mL) Albumin 10(mg/mL) 1. Autocolle ® and Biocoll ® are from Centre de TransfusionSanguine de Lille (Tours, France) 2. Tissucol ® is from Immuno AG(Vienna, Austria) 3. Beriplast ® is from Behringwerke (Marburg, Germany)4. Transglutine ® is from CNTS Franctionation Centre (Strasbourg,France)

Based on the above-described studies, it was established thatapproximately 40 mg fibrin glue to approximately 1000 mg β-TCP yields animproved device as contemplated herein. Based on the same studies, itwas established that approximately 20 mg fibrin glue to approximately1000 mg collagen yields a device with the preferred properties set forthherein. Generally, the data suggest that a range of approximately 20-220mg fibrin glue/1000 mg matrix can maximize cohesiveness, integrity andhandling properties, depending on the precise circumstances and intendeduse.

IV. Other Materials Considerations

In certain embodiments of the invention, the preferred matrix materialis β-TCP. Preferred characteristics of a non-synthetic, non-polymericmaterial for use as a matrix in the claimed invention include, but arenot limited to: a high rate of resorption of the matrix by thesurrounding tissue and a low inflammatory response. As discussed above,sintered, high fired β-TCP having particle sizes ranging from about 212μm to about 425 μm are currently most preferred, but other particlesizes can be used to practice the instant invention.

Image Analysis Method

The rate of resorption of the β-TCP matrix was determined using astandard image analysis method. Image analysis is a method of evaluatingthe particle size distribution of Ca/P granules. The particle size ofCa/P granules is compared before and after implantation in rats. Thesoft tissues of the explants are dissolved by sodium hypochlorite, andthe remaining Ca/P granules are washed several times with water anddried at room temperature. The particles are mixed with glycerol andmounted onto glass slides. Particle size is determined by microscopyusing a standard image analysis system, such as Bioquant OS/2 linked bya video camera to the microscope. Arrays denoting the area and thelongest diameter are selected to express the individual particledimensions. Image of the particles showing gray scale of 0 to 88 on a256-level set were chosen and measured. The raw data from individualparticles are used to calculate the mean and standard deviation. Atleast 50 particles are measured in each data set.

Rat Sub-Cutaneous Study

In the study set forth below, β-TCP (Clarkson, #211096, BD=0.86, 212-425μm, 9/6/97) is formulated in a CMC/blood paste with or without 10 μgOP-1 and implanted into rat subcutaneous sites. The implants are removedafter 6 and 12 weeks in vivo and analyzed using the image analysismethod described above. Results at six weeks are summarized in Table 4Dbelow. Results indicate that after six weeks the size of β-TCP decreasesfrom 334 μm to 184 μm (without OP-1) and to 166 μm (with OP-1). Thedifference in size between OP-1 treated and non-treated samples is notsignificant. However, there is about 50% reduction in the diameter ofthe β-TCP after six weeks.

TABLE 4D IN VIVO RESORPTION OF β-TCP AT 6 WEEKS time in OP-1 Particlesize Particle area Samples (n = 4) vivo (μg) (μm) (mm²) β-TCP/CMC/Blood6 weeks  0 184 +/− 29 0.022 +/− 0.008 β-TCP/CMC/Blood 6 weeks 10 166 +/−22 0.016 +/− 0.004 β-TCP alone 0 — 334 +/− 16 0.068 +/− 0.007 (212-425μm)

TABLE 4E Source and Composition of Some Preferred Matrix Materials andPreferred Components of Fibrin Glue. Item Source Composition Ca/Pparticles Clarkson Chromatography Range from 50-2000 μm, Products, Inc.for example, hydroxyapatite (S. Williamsport, PA) (Ca₁₀(PO₄)₆(OH)₂) orβ-TCP (Ca₃(PO₄)₂) Bovine Sigma (F8630) 75% protein, 10% sodiumFibrinogen (St. Louis, MO) citrate, 15% NaCl Human Immuno AG 500 U or 4Uper vial, Thrombin (Vienna, Austria) reconstituted in 2 mL 40 mM CaCl₂β-TCP granules Clarkson Chromatography Range from 50-2000 μm, Products,Inc. composition is (S. Williamsport, PA) (Ca₃(PO₄)₂) Calf serum LifeTechnologies n.a. (16170-078) (Gaithersburg, MD) bovine Sigma (T4648)50-100 U/mg protein thrombin (St. Louis, MO) Rat thrombin Sigma (T5772)1000 U/mg protein (St. Louis, MO) Rat Fibrinogen Sigma (F6755) 70%protein, 12% sodium (St. Louis, MO) citrate, 18% NaCl

Inflammation

In general, it can be assumed that small particles will be resorbedfaster than large particles. Results indicated that, without OP-1, β-TCP(212-425 μm) elicited a slightly elevated inflammatory response.However, the inflammatory reaction was reduced as the dose of OP-1increases from 10 μg to 20 μg. Previous animal studies have shown thatsmall particles, less than 10 μm elicit a high inflammatory response.Therefore, use of sintered p-TCP (100%) particles of the size of 212 to425 μm is a balance between resorption rate, low inflammation, andability to support bone formation in the rat subcutaneous model.

V. Bioassay A. Bioassay of Osteogenic Activity: Endochondral BoneFormation and Related Properties

The following sets forth exemplary protocols for identifying andcharacterizing bona fide osteogenic or bone morphogenic proteins as wellas osteogenic devices within the scope of Applicants' invention.

The art-recognized bioassay for bone induction as described by Sampathand Reddi (Proc. Natl. Acad. Sci. USA (1983) 80:6591-6595) and U.S. Pat.No. 4,968,590, the disclosures of which are herein incorporated byreference, is used to establish the efficacy of the purificationprotocols. Briefly, this assay consists of depositing the test samplesin subcutaneous sites in allogenic recipient rats under etheranesthesia. A vertical incision (1 cm) is made under sterile conditionsin the skin over the thoracic region, and a pocket is prepared by bluntdissection. In certain circumstances, approximately 25 mg of the testsample is implanted deep into the pocket and the incision is closed witha metallic skin clip. The heterotropic site allows for the study of boneinduction without the possible ambiguities resulting from the use oforthotopic sites.

The sequential cellular reactions occurring at the heterotropic site arecomplex. The multistep cascade of endochondral bone formation includes:binding of fibrin and fibronectin to implanted matrix, chemotaxis ofcells, proliferation of fibroblasts, differentiation into chondroblasts,cartilage formation, vascular invasion, bone formation, remodeling, andbone marrow differentiation.

In rats, this bioassay model exhibits a controlled progression throughthe stages of matrix induced endochondral bone development including:(1) transient infiltration by polymorphonuclear leukocytes on day one;(2) mesenchymal cell migration and proliferation on days two and three;(3) chondrocyte appearance on days five and six; (4) cartilage matrixformation on day seven; (5) cartilage calcification on day eight; (6)vascular invasion, appearance of osteoblasts, and formation of new boneon days nine and ten; (7) appearance of osteoblastic and bone remodelingon days twelve to eighteen; and (8) hematopoietic bone marrowdifferentiation in the ossicle on day twenty-one.

Histological sectioning and staining is preferred to determine theextent of osteogenesis in the implants. Staining with toluidine blue orhemotoxylin/eosin clearly demonstrates the ultimate development ofendochondral bone. Twelve day bioassays are sufficient to determinewhether bone inducing activity is associated with the test sample.

Additionally, alkaline phosphatase activity can be used as a marker forosteogenesis. The enzyme activity can be determinedspectrophotometrically after homogenization of the excised testmaterial. The activity peaks at 9-10 days in vivo and thereafter slowlydeclines. Samples showing no bone development by histology should haveno alkaline phosphatase activity under these assay conditions. The assayis useful for quantitation and obtaining an estimate of bone formationvery quickly after the test samples are removed from the rat. Forexample, samples containing osteogenic protein at several levels ofpurity have been tested to determine the most effective dose/puritylevel, in order to seek a formulation which could be produced on anindustrial scale. The results as measured by alkaline phosphataseactivity level and histological evaluation can be represented as “boneforming units”. One bone forming unit represents the amount of proteinthat is needed for half maximal bone forming activity on day 12.Additionally, dose curves can be constructed for bone inducing activityin vivo at each step of a purification scheme by assaying variousconcentrations of protein. Accordingly, the skilled artisan canconstruct representative dose curves using only routine experimentation.

B. Cartilage Formation: Immunohistochemistry Histology and PolarizedLight Microscopy

1. Immunohistochemistry and Histology.

Briefly, it is well known in the art that identification of bona fidearticular cartilage can be accomplished using ultrastructural and/orbiochemical parameters. For example, articular cartilage forms acontinuous layer of cartilage tissue possessing identifiable zones. Thesuperficial zone is characterized by chondrocytes having a flattenedmorphology and an extracellular network which does not stain, or stainspoorly, with toluidine blue, indicating the relative absence ofsulphated proteoglycans. Toluidine blue is commonly used for thestaining of bone and cartilage. It is a metachromatic stain that yieldsdifferent colors based on the presence of densely spaced negativecharges in the tissues leading to the aggregation and polymerization ofthe dye which shifts the color from blue to purple. Bone is stained bluewhereas the cartilage, with its acidic mucopolysaccharides, is stained adark purple. Chondrocytes in the mid and deep zones have a sphericalappearance, and the matrix contains abundant sulphated proteoglycans, asevidenced by staining with toluidine blue. Collagen fibers are presentdiffusely throughout the matrix. The chondrocytes possess abundant roughendoplasmic reticulum and are surrounded by an extracellular network.The pericellular network contains numerous thin, non-banded collagenfibers. The collagen in the interterritorial network is less compactedand embedded in electron translucent amorphous material, similar toarticular cartilage. Collagen fibers in the interterritorial region ofthe network exhibit the periodic banding characteristic of collagenfibers in the interterritorial zone of cartilage tissue.

Von Kossa staining shows a dense black staining of the mineralizedtissue This stain clearly depicts the existing and newly regeneratedbone through the deposition of silver on the calcium salts. Typically,the counter stain is Safranin O, which stains the cartilage red-orange.New and existing bone can usually be easily distinguishedmorphologically in sections stained accordingly. Safranin O/Fast Greenis able to distinguish more features than the Toluidine blue. Safranin Ois a basic dye that stains the acidic mucopolysaccharides in thearticular cartilage red-orange and the underlying subchondral bone onlylightly. Fast Green is an acidic dye that stains the cytoplasmgray-green. Stain is not only able to clearly identify the existing andregenerated cartilage, but can also distinguish differences between tworegions in the reparative tissue indicating differences in the contentof proteoglycans.

Hematoxylin/eosin stains which depict bone a darker red and thecarbohydrate rich cartilage only very lightly, can also be used. MassonTrichrome is able to distinguish differences in the reparative tissue.Cartilage and acidic polysaccharide-rich reparative tissue, muscle, anderythrocytes are stained red, with the collagen of the bone stainedblue.

Histological evaluations can also involve assessment of:glycosaminoglycan content in the repair cartilage; cartilage andchondrocyte morphology; and, structural integrity and morphology at thedefect interface. The morphology of repair cartilage can be identifiedby the type of cartilage formed: articular vs. fibrotic by evaluatingglycosaminoglycan content, degree of cartilage deposition, and the like.

Histological evaluations using standard methodologies well characterizedin the art also allows assessment of new bone and bone marrow formation.See, for example, U.S. Pat. No. 5,266,683, the disclosure of which isincorporated herein by reference.

Additionally, it is well known in the art that biochemically, thepresence of Type. II and Type IX collagen in the cartilage tissue isindicative of the differentiated phenotype of chondrocytes. The presenceof Type II and/or Type IX collagen can be determined by standard gelelectrophoresis, Western blot analysis and/or immunohisto-chemicalstaining using, for example, commercially available antibody asdescribed below. Other biochemical markers include hematoxylin, eosin,Goldner's Trichrome and Safranin-O.

Immunohistochemical methods, such as the following, can be utilized toidentify formation of cartilage tissue, including articular cartilage.Tissue sections are prepared using routine embedding and sectioningtechniques known in the art. Epitopes for Type II collagen are firstexposed by protease pretreatment. For example, tissue specimens arepretreated with 1 mg/ml pronase type XIV from Sigma (St. Louis, Mo.;catalog number. P5147) in tris-buffered saline (TBS) for approximately10 min at room temperature. Specimens are then washed in TBS with 0.2%glycine. Specimens are blocked for 30 min, in a tris-buffered salinesolution containing 1% Tween 20 (TBST) and bovine serum albumin (BSA),and washed with TBST. Specimens are then incubated with affinitypurified polyclonal goat anti-human collagen Types I and II antibodiesfor approximately 1 hr, or overnight, at room temperature. In certain ofthe Examples set forth below, goat anti-human Type I collagen antibodywas obtained from Southern Biotechnology Associates (Birmingham, Ala.),catalog number 1310-01, for example, lot number L055-X916; goatanti-human Type II collagen antibody was also obtained from SouthernBiotechnology Associates, catalog number 1320-01, for example, lotnumber C153-T826. Anti-human Types I and II collagen antibodiesgenerated in mouse or rabbit can also be used. The skilled artisan willappreciate the circumstances under which use of one species versusanother is appropriate. For certain of the examples set forth below, theconcentrations of goat anti-human Types I and II collagen antibodiesused for incubation is, for example, 20 μg/ml for each antibody dilutedinto 1% BSA in TBST. After incubation with antibodies, the specimens arerinsed with TBST and held in a bath. A commercially available linkantibody is then added. For example, specimens treated with goatanti-human collagen Types I and II antibodies can be incubated withgoat-link antibody from BioGenex Laboratories (San Ramon, Calif.);catalog number HK209-5G) for at least 10 min at room temperature. Forthose samples incubated with mouse or rabbit antibodies, a Dako LSAB2kit number K0610 from Dako Corporation (Carpinteria, Calif.) can be usedas the link antibody. The specimens are again rinsed with TBST and heldin a bath. Next, the specimens are allowed to incubate withStrepavidin/Alkaline Phosphatase commercially available from any of theabove-identified sources for at least approximately 10 min at roomtemperature. The specimens are again rinsed with TBST. The specimens arethen developed by treatment with an appropriate substrate solution forapproximately 10 min or less. For example, for alkaline phosphatasedetection, approximately 100 μl of 50× lavamesole is used. For colordevelopment, Fast Red from Dako Corporation is used. After development,the specimens are counterstained by washing for 2 min with Harrishematoxylin and 1% lithium carbonate. The specimens are then mounted inan aqueous mounting media, and cartilage formation is subsequentlyevaluated.

Staining for types I and II collagen is useful to determine the boundarybetween regenerated subchondral bone and reparative tissue. Generally,reparative tissue that is fibrous stains less intensely. Additionally,newly formed subchondral bone can be identified by type II collagenlocalization in small spicules of remnant cartilage. Toluidene blue andSafranin-O are also useful for staining acidic proteoglycans in acartilage layer as well as reparative tissues.

2. Polarized Light Microscopy

Polarized light microscopy can be used to assess fibril interdigitationat the junction between the margins of repair tissue and the residualarticular cartilage adjacent to the defect. Such microscopy can beperformed using Safranin-O stained sections from a defect. In certaininstances, polarized light microscopy offers the skilled artisan a moreaccurate view of the repair process. For example, using lightmicroscopy, repair tissue at the periphery of a defect can appear wellapposed with the residual cartilage. Using polarized light microscopy,however, it can be observed that the collagen fibrils of the repairtissue are not well integrated with those of the residual cartilage.Lack of fibril continuity between repair and persisting cartilage isindicative of sub-optimal repair.

Thus, when evaluating qualitatively the interface between repaircartilage and residual viable cartilage, fibrillar continuity ispreferably assessed using polarized light microscopy as exemplifiedherein below. (See, also, Shapiro et al., Journal of Bone and JointSurgery 532-553 (1993), the disclosure of which is herein incorporatedby reference.)

Practice of the invention will be still more fully understood from thefollowing examples, which are presented herein for illustration only andshould not be construed as limiting the invention in any way.

VI. Animal Studies: Methods of Use of Improved Osteogenic Devices A.Repair of Critical Size Segmental Defects Using Improved OsteogenicDevices Containing Carboxymethylcellulose

1. Experiment 1: Unitary Device Configuration (Dogs)

This study illustrates the efficacy of OP-1 combined with collagenmatrix and carboxymethylcellulose for repairing critical-size ulnasegmental defects in the art-recognized canine model.

Briefly, the data set forth below indicate at least comparableradiographic healing at sites that received a CMC/OP-1 device relativeto segmental defects treated with the standard OP device. The finalradiographic grade (maximum=6.0) for defects treated with CMC/OP-1 was5.33±0.58 compared to 4.67±0.58 for defect receiving the standard OP-1device. In general, new bone formation was evident as early as two weekspost-operative in all defects. The new bone continued to densify,consolidate and remodel until sacrifice at twelve post-operative weeks.The mean load to failure of the defects treated with the CMC/OP-1 devicewas 59.33 N±26.77. This was 70% of the mean load to failure of thecontralateral sides which received the standard OP-1 implants.Histologically, the final volume, quality and degree of remodelling wereat least equivalent in defects treated with the CMC/OP-1 and standardOP-1 device, although a variation in the final new bone formation anddegree of remodelling was noted in animal to animal comparisons. Themean histologic grade for defects treated with the CMC/OP-1 device was12.67±1.04 out of 16 total possible points. The mean histologic gradefor defects treated with the standard OP-1 device was 11.41±0.95 out of16 total possible points.

Test Device Description

As already described, standard devices consisted of recombinant humanosteogenic protein-1 (rhOP-1) admixed with bovine bone Type I collagenmatrix at a ratio of 2.5 mg rhOP-1 per gram of collagen matrix. Theimproved device consisted of rhOP-1 admixed with bovine bone Type Icollagen matrix and carboxymethylcellulose (CMC). The unitary deviceswere supplied in sterile vials.

As earlier-described, the currently preferred CMC-containing device foropen defects has a putty consistency. The unitary CMC/OP-1 device wasplaced dry into a small bowl and mixed with saline. Using fingers, thepractitioner mixed and formed the device into the general shape of thedefect and then placed the device into the defect site. It was reportedthat the improved device was more easily handled and shaped, and did notstick to the surgical gloves. The device maintained its integrity whenplaced in the defect during irrigation and during/after suturing.

Experimental Design

Adult male mongrel dogs were utilized because of their well-known bonerepair and remodeling characteristics. All animals were at least twoyears old and weighed from 40 to 50 pounds. All animals were supplied byMartin Creek Kennels, USDA number 71-B-108, Willowford, Ak. Specialattention was paid in selecting animals of uniform size and weight tolimit the variability in bone geometry and loading. The animals wereradiographically screened pre-operatively to ensure proper size,skeletal maturity, and that no obvious osseous abnormalities existed.

A total of 3 adult male dogs were utilized. Bilateral 2.5 cm ulnasegmental defects were created. All right side defects received theimproved device (CMC/OP-1 device). All left side defects receivedstandard OP-1 device. Biweekly radiographs were taken to study theprogression of healing and graded on a 0-6 scale. At sacrifice, allulnae were retrieved en bloc, and those that were healed sufficientlyupon manual manipulation were mechanically tested in torsion. Segmentswere evaluated by histology for tissue response, bone architecture andremodelling, and quality and amount of new bone formation and healing;grading was on a 0-16 scale.

Surgery

Using standard aseptic techniques, surgery was performed under halothanegas anesthesia. A lateral incision approximately 4.0 cm in length wasmade and exposure of the ulna was obtained using blunt and sharpdissection. A 2.5 cm segmental osteoperiosteal defect was created in themid-ulna using an oscillating saw. This defect was about 2-2.5 times themid-shaft diameter, and represents a critical size defect, i.e., thedefect would not heal spontaneously. Intra-operative measurements weremade of the removed bone segment. The radius was maintained formechanical stability, but no internal or external fixation was used. Thesite was irrigated with saline to remove bone debris and spilled marrowcells. After the site was dried and homeostasis was achieved, theimplants were carefully placed into the defects. The soft-tissues weremeticulously closed in layers to contain the implant. The procedure wasthen repeated on the contralateral side.

Radiographs

Radiographs of the forelimbs were obtained biweekly until eight weekspost-operative and then again at sacrifice at twelve post-operativeweeks. Standardized exposure times and intensities were used, andsandbags were used to position the extremities in a consistent manner.Radiographs were evaluated and compared to earlier radiographs toappreciate quality and speed of defect healing. Grading of radiographswas in accordance with the following scale:

TABLE 5 Radiographic Grading Scale Grade: No change from immediatepost-operative appearance 0 Trace of radiodense material in defect 1Flocculent radiodensity with flecks of calcification 2 Defect bridged atleast one point with material of 3 non-uniform radiodensity Defectbridged on both medial and lateral sides 4 with material of uniformradiodensity, cut end of the cortex remain visible Same as grade 3; atleast one of four cortices is 5 obscured by new bone Defect bridged byuniform new bone; cut ends of 6 cortex are no longer distinguishable

Sacrifice

At the end of the study period, animals were sacrificed using anintravenous barbiturate overdose. The ulna and radius were immediatelyharvested en bloc and placed in saline soaked diapers. Both ulna weremacrophotographed and contact radiographs with labels were taken. Softtissues were carefully dissected away from the defect site. Awater-cooled saw was used to cut the ulna to a uniform length of 9 cmwith the defect site centered in the middle of the test specimen.

Mechanical Testing

Immediately after sectioning, if healing was deemed sufficient by manualmanipulation, specimens were tested to failure in torsion using routineprocedures on an MTS closed-loop hydraulic test machine (Minneapolis,Minn.) operated in stroke control at a constant displacement rate of 50mm/min. Briefly, each end of the bone segment was mounted in acylindrical aluminum sleeve and cemented with methylmethacrylate. Oneend was rigidly fixed and the other was rotated counterclockwise. Sincethe dog ulna has a slight curvature, the specimens were mounted to keepspecimen rotation coaxial with that of the testing device. The torsionalforce was applied with a lever arm of 6 cm, by a servohydraulicmaterials testing system. Simultaneous recordings were made of implantdisplacement, as measured by the machine stroke controller, while loadwas recorded from the load cell. Force-angular displacement curves weregenerated from which the torque and angular deformation to failure wereobtained, and the energy absorption to failure computed as the areaunder the load-displacement curve.

Histology

The individual specimens were fixed by immersion in 10% bufferedformalin solution immediately following mechanical testing or aftersectioning in untested specimens. On a water cooled diamond saw thespecimens were divided by bisecting the specimen down its long axis.This procedure, resulted in two portions of each specimen for differenthistologic preparations, including undecalcified ground sections andundecalcified microtome sections.

Following fixation, the specimens designated for undecalcified sectionswere dehydrated in graduated ethyl alcohol solutions from 70% to 100%.The specimens were then placed in methylmethacrylate monomer and allowedto polymerize. The ground sections were obtained by cutting thespecimens on a high speed, water cooled Mark V CS600-A (Grandby, Conn.)sectioning saw into sections approximately 700 to 1,000 μm thick.Sections were mounted on acrylic slides and ground to 100 μm thicknessusing a metallurgical grinding wheel, and microradiographs were madeusing standardized techniques. Following microradiography, the sectionswere further ground to approximately 50 μm and stained with basicfuchsin and toluidine blue for histologic grading that evaluated thefollowing parameters of repair: quality of the union, the appearance andquality of the cortical and cancellous bone, the presence of bone marrowelements, bone remodelling, and inflammatory response. Grading ofhistologic parameters was in accordance with the following scale:

TABLE 6 Histologic Grading Scale Grade: Quality of Union: no sign offibrous or other union 0 fibrous union 1 osteochondral union 2 boneunion 3 bone union with reorganization of cortices 4 Cortex Development:none present in the defect 0 densification of borders 1 recognizableformation 2 intact cortices but not complete 3 complete formation ofnormal cortices 4 Residual Implant Material/Internal Architecture: largeamounts of implant material visible 0 moderate amount of residualimplant material 1 small amount of residual implant 2material/unorganized architecture no residual implant/return of marrow 3cavity/some marrow elements normal marrow elements and architecture 4Inflammatory Response: severe 0 severe/moderate 1 moderate response 2mild 3 no response 4 TOTAL POINTS 16

Results Radiographic Evaluation

In this study, there were no significant differences in the radiographicbone healing characteristics of sites that received a CMC/OP-1 device ascompared to segmental defects treated with the standard OP device. Ingeneral, new bone formation was evident as early as two weekspost-operative in all defects. The new bone continued to densify,consolidate and remodel until sacrifice at twelve post-operative weeks.New cortex development with early medullary cavity formation occurredbetween the 6 and 8 week evaluations. The final radiographic grade fordefects treated with the CMC/OP-1 device was 5.33±0.58. The finalradiographic grade for defects receiving the standard OP-1 device was4.67±0.58.

As an example, specific representative observations for one of the testanimals are set forth below:

Right Defect (CMC/OP-1 Device)

At two weeks post-operative, traces of radiodense material were presentin the right defect, but, the defect was not completely bridged orfilled with new bone. By four weeks post-operative, the amount andradiodensity of the new bone significantly increased. The defect wasspanned, but, the new bone was not well contained. There was someconsolidation of new bone along the periosteal borders. An equivalentamount of new bone had formed compared to the left defect of thisanimal. At six weeks post-operative, the radiodensity of the new boneincreased and the defect was completely spanned and filled withextensive new bone. Early remodelling was evident with the host boneends beginning to incorporate with the new bone. At eight weekspost-operative, new bone continued to remodel and the new bone volumebetter approximated the defect borders. The host bone ends wereincorporated with the new bone with densification of new bone along theborders suggestive of new cortex formation. There was no radiographicevidence of any residual carrier material. At sacrifice a radiolucentregion was present within the center of the right side defect, but,densification of the new bone borders was suggestive of new cortexformation. The final radiographic grade was 5 out of 6 possible points.

Left Defect (OP-1 Device)

At two weeks post-operative, traces of radiodense material were presentin the defects, but, the defect was not bridged or filled with new bone.At four weeks post-operative, the amount and radiodensity of the newbone significantly increased and the defect was spanned and filled withnew bone. At six weeks post-operative, the radiodensity of the new boneincreased and the defect was completely spanned and filled withextensive new bone. Early remodelling was evident, and the new bonevolume better approximated the defect borders. The new bone wasuniformly dense, and the host bone ends were beginning to incorporate.At eight weeks post-operative new bone continued to remodel, the hostbone ends were incorporated, and densification of the new bone had begunalong the defect borders. At sacrifice densification of the new bonealong the defect borders was suggestive of early reformation of newcortices. The density of the new bone within the center of the defectwas greater than new bone that had formed in the right side defect,although there were no significant differences in the radiographicappearances of the right and left sides in this animal. The finalradiographic grade was 5 out of 6 possible points.

Gross Observations

Both right and left specimens from all animals had similar grossappearances. In two animals, the right and left defects were firmlyunited and had approximately the same volume of new bone. In a thirdanimal, both the right and left side had a similar new bone volume, but,the left side was not completely united.

Mechanical Testing

The mean load to failure for defects treated with the CMC/OP-1 devicewas 59.33 N±26.77 (n=3). The mean load to failure was 79% of the meanload to failure of the contralateral sides, which received the standardOP-1 devices. This represented 91% of the strength of intact controlstested previously. The mean angular deformation was 38.22±0.69 degrees.The mean energy absorbed to failure was 97.47±47.21 Nm degrees.

The mean load to failure of the defects treated with the standard OP-1device was 75.39 N±1.88 (n=2). This represented 115% of the strength ofintact controls tested previously. The mean angular deformation was59.06±27.80 degrees. The mean energy absorbed to failure was 93.40±17.49Nm degrees. As noted, one defect treated with the standard OP-1 devicewas not tested due to gross instability.

Histology

In general, normal bone formation consistent with defects treated withthe standard rhOP-1 device with a collagen matrix were observed. Bothfinal volume and quality and degree of remodelling were equivalent incomparison of CMC/OP-1 and standard OP-1 devices. A variation in thefinal new bone formation and degree of remodelling was noted in animalto animal comparisons. The mean histologic grade for defects treatedwith the CMC/OP-1 device was 12.67±1.04 out of 16 total possible points.The mean histologic grade for defects treated with the standard OP-1device was 11.41±0.95 out of a total of 16 possible points.

Generally, both the left and right defects were spanned by a largevolume of new bone. The new bone was beginning to reorganize and hadlamellar characteristics. Along the defect borders, the new bone becamemore dense and was suggestive of new cornices. In certain instances,remodelling was not as uniformly advanced on the left as the right side.The volume of new bone on the left side was slightly less than on theright in certain instances. At the center of all the defects, the returnof medullary components was evident.

In conclusion, improved osteogenic devices (unitary configuration) wereused to repair critical size segmental defects. Rates of endochondralbone repair, mechanical strength indicia, radiographic indicia andhistological indicia suggested improved devices result in defect repairat least comparable to standard osteogenic devices.

2. Experiment 2: OP-1 Dose Response Using Non-Unitary DeviceConfiguration (Dogs)

This study further illustrates the efficacy of standard osteogenicdevice admixed with carboxymethylcellulose (CMC) using both standard andlow OP-1 dose formulations to heal large, critical size segmentaldefects in the canine ulna segmental defect model.

As described in detail below, various dosages of OP-1 were employed inthis study. Briefly, the low dose formulations of the OP-1 devicewithout CMC were found effective at inducing new bone formation, butless so than the standard dose OP-1 device. However, and unexpectedly,defects treated with the low dose CMC-containing device demonstratedearlier and larger volumes of new bone formation compared to the lowdose OP-1 device without CMC. The standard or low dose OP-1 device wasprepared by combining a 1 g OP-1 device with 3.2 ml sterile saline. Thestandard or low dose OP-1 device containing CMC was prepared bycombining 1 g OP-1 device with 0.2 g CMC and approximately 2 ml sterilesaline. The devices were prepared intra-operatively. Radiographically,standard dose OP-1 treated sites with and without CMC had similarradiographic appearances. Standard dose OP-1 sites had earlier andgreater volumes of new bone formation compared to the low dose sites.Histologic results demonstrated more advanced segmental bone defecthealing in sites treated with the CMC-containing device compared to thestandard OP-1 device. Sites treated with low dose OP-1 device containingCMC achieved an equivalent degree of remodeling and incorporation withthe host bone relative to sites treated with the standard dose OP-1device, but, the volume of new bone induced was less. Defect sitestreated with standard dose device containing CMC obtained the greatestmean torsional load to failure at twelve weeks post-operative comparedto all other treatment groups (61.91±35.37 N, 95% of the torsionalstrength of intact controls). The torsional strength of the low dosedevice containing CMC sites was similar to the standard OP-1 device,having 78% of the strength of intact ulnae and 99% of the strength ofpreviously tested sites treated with the standard device. In contrast,the torsional strength of the low dose OP-1 sites without CMC was only44% of the torsional strength of intact ulnae and 56% of previouslytested segmental defects treated with the standard OP-1 device.

Test Material

The standard OP-1 device (designated OP in Table 11) consisted ofrecombinant human osteogenic protein-1 (rhOP-1) admixed with bovine boneType I collagen matrix at a ratio of 2.5 mg rhOP-1/g of collagen matrix.One CMC device (standard dose, designated OP-1/CMC, OPCMC in Table 11)consisted of an OP-1 device combined with carboxymethylcellulose. Thelow dose OP-1 device consisted of 1.25 mg rhOP-1/g of collagen matrix(designated LOP in Table 10). Each OP-1 device in both standard and lowdose consisted of 1 g of device packaged separately from CMC. CMC waspackaged 200 mg per vial. This is in contrast to the unitary devicedescribed above wherein CMC was co-packaged with the other components ofcollagen matrix and osteogenic protein.

Experimental Design

A total of 12 adult mongrel dogs were utilized. Bilateral 2.5 cmcritical size ulna segmental defects were created. The right sidedefects of six animals received the standard OP-1 device. The left sidedefects of this group received the standard dose OP-1/CMC devices. Thesecond group of six animals received the low dose OP-1 devices in theright side defect and received low dose OP-1/CMC devices in the leftside defects. Biweekly radiographs were taken to study the progressionof healing. At sacrifice, all were retrieved in bloc and mechanicallytested in torsion. Ulna segments were evaluated by histology for tissueresponse, residual implant, and quality and amount of new bone formationand healing.

Animal Model

As described above, adult male mongrel dogs were utilized because oftheir anatomical size, and known bone repair and remodelingcharacteristics. All animals were skeletally mature and weighed from 35to 50 pounds.

Surgery

Using standard surgical techniques similar to those described above, alateral incision approximately 4 cm in length was made and exposure ofthe ulna was obtained using blunt and sharp dissection. A 2.5 cmsegmental osteoperiosteal defect was created in the mid-ulna using anoscillating saw. This defect was about 2-2.5 times the mid-shaftdiameter, and represented a critical-sized defect, i.e., the defectwould not heal spontaneously. Intra-operative measurements were made ofthe removed bone segment. The length of the segment, the two outerdiameters of the segment, and the central diameter of the segment wasrecorded in millimeters in the surgical records. The radius wasmaintained for mechanical stability. The site was irrigated with salineto remove bone debris and spilled marrow cells. After the site was driedand homeostasis was achieved, the implants were placed in the defect.The soft-tissues were closed in layers to contain the implant. Theprocedure was then repeated on the contralateral side.

Radiographs

As described above, radiographs of the forelimbs were obtained biweeklyuntil eight weeks post-operative and then again at sacrifice at twelvepost-operative weeks.

Sacrifice

Procedures were similar to these described above. At the end of thestudy period, animals were sacrificed, the ulna and radius immediatelyharvested en bloc and placed in saline soaked diapers. Soft tissues werecarefully dissected away from the defect site. A band saw was used tocut the ulna to a uniform length of 9 cm with the defect site centeredin the middle of the test specimen.

Mechanical Testing

Protocols were similar to those described above. Briefly, specimens weretested to failure in torsion on an MTS closed-loop hydraulic testmachine (Minneapolis, Minn.) operated in stroke control at a constantdisplacement rate of 50 mm/min. Torsional force was applied with a leverarm of 6 cm, by a servohydraulic materials testing system. Simultaneousrecordings were made of implant displacement, as measured by the machinestroke controller, while load was recorded from the load cell.Force-angular displacement curves were generated from which the torqueand angular deformation to failure were obtained, and the energyabsorption to failure was computed as the area under theload-displacement curve.

Histology

As described above, fixed specimens designated for undecalcifiedsections were dehydrated in graduated ethyl alcohol solutions from 70%to 100%. The specimens were then placed in methylmethacrylate monomerand allowed to polymerize. The ground sections were obtained by cuttingthe specimens on a high speed, water cooled sectioning saw into sectionsapproximately 700 to 1,000 μm thick. These sections were mounted onacrylic slides and ground to 100 μm thickness. Following routinemicroradiography, the sections were further ground to approximately 50μm and stained with basic fuchsin and toluidine blue for histologicgrading that evaluated the following parameters of repair: quality ofthe union, the appearance and quality of the cortical and cancellousbone, the presence of bone marrow elements, bone remodeling, andinflammatory response.

Radiographic Evaluation

At twelve weeks post-operative (sacrifice), the standard dose OP-1/CMCsites achieved the greatest mean radiographic grade, 5.17/6.0 points.The final radiographic grade for the standard OP-1 devices was 5.00/6.0.The low dose OP-1 sites had a mean final radiographic grade of 3.83/6.0.Low dose OP-1/CMC sites had a mean grade of 4.67/6.0. At all timeperiods the standard dose OP-1/CMC sites had greater mean radiographicgrades than standard OP-1 without CMC. At all time periods the low doseOP-1 CMC sites had greater mean radiographic grades than low dose OP-1without CMC sites.

Statistical analysis demonstrated a significant effect for implant typewhen all radiographic grades were combined (Kruskal-Wall is one wayanalysis of variance, p=0.0049). Multiple comparisons demonstrated thatthe standard OP-1 and the standard dose OP-1/CMC devices meanradiographic grades for all time periods were significantly greater thanthe low dose OP-1 sites without CMC (at α=0.10 and at α=0.05,respectively). Multiple comparisons also demonstrated that the meanradiographic grade of the standard dose OP-1/CMC sites was significantlygreater than the low dose OP-1/CMC sites (α=0.10). However, andunexpectedly, the standard dose OP-1 devices mean radiographic gradeswere not significantly greater than the mean radiographic grades for thelow dose OP-1/CMC sites.

Low Dose OP-1 Sites without CMC

At two weeks post-operative, new bone formation was evident in one ofsix defects treated with low dose OP-1. Traces of radiodense materialwere present around the defect, but new bone did not bridge or span thedefect. The mean radiographic grade at two week post-operative was0.17/6.0 points. At four weeks post-operative, the same sitedemonstrating new bone formation at two weeks demonstrated an increasein new bone volume. Four defects were spanned and one defect was filledwith new bone at four weeks. Two sites demonstrated little activity atfour weeks post-operative. The mean radiographic grade at four weeks was1.83/6.0. By six weeks post-operative, two sites treated with low doseOP-1 were spanned and filled with new bone. Two sites were spanned butincompletely filled with new bone. One animal demonstrated some earlynew bone formation. One animal did not demonstrate any new boneformation. The mean radiographic grade at six weeks was 2.83/6.0. Fromsix to eight weeks additional new bone formation was not evident, but,some densification of new bone was apparent and some early remodelinghad occurred. The mean radiographic grade at eight weeks was 3.17/6.0.At sacrifice, twelve weeks post-operative, all defects demonstrated somewell contained new bone, but, the density was significantly less thanthe surrounding host bone. Occasional radiolucencies at the host bonenew bone junction were present. The mean radiographic grade at twelveweeks was 3.83/6.0.

Low Dose OP-1/CMC Sites

At two weeks post-operative, early new bone formation was evident inthree of six defects treated with low dose OP-1/CMC. New bone did notspan or fill the defects, but was well contained within the surgicalsites. The mean radiographic grade at two weeks was 0.83/6.0. At fourweeks post-operative, new bone formation was present in five of sixdefects, spanning and almost filling the defects. The mean radiographicgrade at four weeks was 2.33/6.0. At six weeks the density of new bonepresent in the defects increased. Early incorporation of the host bonewas evident in three of six defects. One animal did not demonstrate anynew bone formation bilaterally at six weeks. The mean radiographic gradeat this time was 3.00/6.0. From six to eight weeks, no additional newbone formation occurred. Early remodeling and incorporation with thehost bone was apparent. One animal did not demonstrate any changes inradiographic appearance. The mean radiographic grade at eight weeks was3.33/6.0. Unexpectedly, the low dose OP-1/CMC sites demonstrated moreextensive new bone formation and remodeling than the low dose OP-1 siteswithout CMC. In sites where the defect was completely filled with newbone, the density of the new bone was less than the surrounding hostbone. The mean radiographic grade at twelve weeks (sacrifice) was4.67/6.0.

Standard OP-1 Device Sites

The results in this study were consistent will all previous studies ofthe standard OP-1 device. At two weeks post-operative, four of sixdefects treated with the OP-1 device demonstrated early new boneformation. In two defects, extensive new bone spanned the defects, but,new bone did not fill the defects. Overall, the new bone was not wellcontained. The mean radiographic grade at two weeks was 1.50/6.0. Atfour weeks post-operative, an increase in the amount and density of newbone occurred in all six defects. New bone spanned all defects. In fourof six sites the defect appeared completely filled with new bone. Themean radiographic grade at four weeks was 3.00/6.0. At six weekspost-operative, the density of new bone increased. New bone was not wellcontained in the remaining defects. Generally, early incorporation atthe host bone ends was observed in three of the six sites. The meanradiographic grade at six weeks was 3.67/6.0. From six to eight weeks,nearly complete incorporation with the host bone was evident in three ofsix sites, although remodeling toward the ulna contours had occurred inall defects. The mean radiographic grade at eight weeks was 4.50/6.0. Bytwelve weeks post-operative (sacrifice) extensive remodeling hadoccurred, although the new bone volume did not yet approximate the ulnacontours. New bone often extended into the surrounding soft tissues,although some reformation of cortices was apparent in all defectstreated with the standard OP-1 device. The mean radiographic grade attwelve weeks was 5.00/6.0.

Standard Dose OP-1/CMC Sites

At two weeks post-operative, early new bone formation was evident infour of six defects treated with OP-1/CMC device. New bone was wellcontained in only one of the four defects. New bone appeared to span andfill two of the six defects. The mean radiographic grade at two weekswas 1.67/6.0. At four weeks post-operative, extensive new bone hadoccurred in all six defects. New bone was not well contained, but, earlyincorporation with the host bone was observed in two sites. The meanradiographic grade at four weeks was 1.67/6.0. From four to six weeks,extensive remodeling and incorporation of the host bone occurred in alldefects. New bone was not well contained, but, new bone in the softtissue had begun to resorb. The mean radiographic grade at six weeks was4.33/6.0. By eight weeks, complete incorporation with the host bone wasappreciated in two sites, and early new cortex formation was evident inat least one site. The mean radiographic grade at eight weeks was4.67/6.0. At twelve weeks post-operative (sacrifice), three of sixdefects had extensive incorporation with the host bone ends. New bonewas still present outside of the defects, although extensive remodelinghad occurred. The mean radiographic grade at twelve weeks was 5.17/6.0.

Gross Observations

Sites treated with low dose implants with and without CMC demonstratedless new bone volume compared to the high dose OP-1 sites with andwithout CMC. All high dose sites were firmly united grossly, but threeof twelve sites treated with low dose OP-1 were not yet firmly united atsacrifice.

Low Dose OP-1 Sites without CMC

In all cases, the amount of new bone formed did not exceed the originaldefect volume. New bone formation was well contained, although in two ofsix segments the bone was not completely united.

Low Dose OP-1/CMC Sites

Similar to defects treated with low dose OP-1, new bone formation waswell contained. One of six sites treated with low dose OP-1/CMC was notcompletely united. Typically, new bone volume was less than the originaldefect volume.

Standard OP-1 Device Sites

Similar to previous studies, new bone volume in sites treated with thestandard OP-1 device was 2 to 3 times greater than the original defectvolume. All defects were firmly united. In five of six defects,extensive new bone extended into the soft tissues and was fused to theradius. In one defect, the volume of new bone formed was less than othersites.

Standard Dose OP-1/CMC Sites

New bone volume in five of six defects treated with OP-1/CMC exceededthe original host bone volume and extended into the soft tissues. Thenew bone volume was 2 to 3 times the volume of the original defect. Asnoted above in one animal, reduced bone volume was observed bilaterally.

Mechanical Testing

Mechanical testing summaries appear in Tables 7 and 8.

Unexpectedly, defect sites treated with the standard dose OP-1/CMCdevice obtained the greatest mean torsional load to failure at twelveweeks post-operative compared to all other treatment groups, includingthe standard device group. The mean load to failure was 61.91±35.37 N(n=6). This represented 95% of the torsional strength of previouslytested intact ulnae and 121% of the strength of previously testedsegmental defects treated with the standard OP-1 device. The standardOP-1 device treated sites had a mean torsional strength of 55.84±37.26 N(n=6), 86% of previously tested intact control ulnae, and 110% ofpreviously tested segmental defects treated with the OP-1 device. Themean load to failure for the low dose OP-1/CMC sites was 50.66±31.68 N(n=5), or 78% of the strength of intact control ulnae and 99% of thestrength of previously tested segmental defects treated with thestandard OP-1 device. The mean load to failure for the low dose OP-1sites was 28.72±14.71 N (n=4). This represented 44% of the torsionalstrength of previously tested intact control ulnae and 56% of previouslytested segmental defects treated with the standard OP-1 device.

Unexpectedly, paired t-tests of the failure load within animalsdemonstrated a significant effect for implant type when comparing lowdose OP-1 standard devices to low dose OP-1/CMC devices (p=0.0597). Thepaired mean load for low dose OP-1 sites was 28.72±14.71 (4). The pairedmean load to failure for the low dose OP-1/CMC sites was 62.89±18.47(4). No significant difference was found in paired t-tests of mean loadto failure standard OP-1 devices compared to the mean load to failurestandard dose OP-1/CMC device.

TABLE 7 Mechanical Testing Results % Intact Energy absorbed AnimalTorque Controls Angulation to failure Number side Implant Type Load toFailure (Nm) (%) (degrees) (NmDegrees) H19 Right LOP 35.17 2.11 53.8836.74 53.93 H21 Right LOP 9.76 0.59 14.95 66.64 16.96 H24 RightLOP * * * * H26 Right LOP 25.75 1.55 39.45 41.33 44.42 H30 RightLOP * * * H34 Right LOP 44.18 2.65 67.69 25.35 41.51 MEAN 28.72 1.7243.99 42.52 39.21 STANDARD 14.71 0.88 22.53 17.43 15.75 DEVIATION SAMPLESIZE 4 4 4 4 4 H19 Left LOPCMC 39.76 2.39 60.92 37.13 55.84 H21 LeftLOPCMC 57.51 3.45 88.11 52.47 59.47 H24 Left LOPCMC 1.74 0.10 2.67 5.540.34 H26 Left LOPCMC 82.33 4.94 126.14 37.85 120.58 H30 LeftLOPCMC * * * * H34 Left LOPCMC 71.94 4.32 110.22 42.47 127.67 MEAN 50.663.04 77.61 35.09 72.78 STANDARD 31.68 1.90 48.54 17.62 52.46 DEVIATIONSAMPLE SIZE 5 5 5 5 5

TABLE 8 Mechanical Testing Results % Intact Energy absorbed AnimalTorque Controls Angulation to failure Number side Implant Type Load toFailure (Nm) (%) (degrees) (NmDegrees) H22 Right OP 65.44 3.93 100.2652.95 139.25 H23 Right OP 2.73 0.16 4.18 15.90 8.91 H25 Right OP 52.783.17 80.86 31.36 70.65 H28 Right OP 27.24 1.63 41.73 53.04 49.49 H32Right OP 79.68 4.78 122.08 19.33 57.83 H33 Right OP 107.14 6.43 164.1529.3 98.59 MEAN 55.84 3.35 85.54 33.65 70.79 STANDARD 37.26 2.24 57.0816.08 44.52 DEVIATION SAMPLE SIZE 6 6 6 6 6 H22 Left OPCMC 76.52 4.59117.24 44.86 150.86 H23 Left OPCMC 6.53 0.39 10.10 68.10 10.66 H25 LeftOPCMC 50.22 3.01 76.94 43.94 69.48 H28 Left OPCMC 100.44 6.03 153.8840.9 177.13 H32 Left OPCMC 43.82 2.63 67.14 40.2 70.58 H33 Left OPCMC93.93 5.64 143.91 43.96 156.09 MEAN 61.91 3.71 94.85 46.99 105.80STANDARD 35.37 2.12 54.19 10.51 65.21 DEVIATION SAMPLE SIZE 6 6 6 5 6*Specimen was not tested

Histology

Unexpectedly, the sites treated with the standard dose OP-1/CMC deviceachieved the greatest mean histologic score, 12.08/16.0 points. The lowdose OP-1/CMC sites achieved a score of 11.07/15.0, slightly greaterthan the mean histologic score for the standard OP-1 device sites,10.88/16.0. The mean histologic grade for the low dose OP-1 sites was9.58/16.0 points.

Statistical analysis of the mean histologic grades by treatment groupdemonstrated a significant effect for implant type (Kruskal-Wallis oneway analysis of variance, p=0.0282). Multiple comparisons of group meansdemonstrated that the mean total grade for the standard dose OP-1/CMCsites was significantly greater than the low dose OP-1 without CMC sites(at α=0.05).

Statistical analysis of the grade for quality of union also demonstrateda significant effect for implant type. Unexpectedly, the mean quality ofunion grade for the standard dose OP-1/CMC sites (3.5/4.0) was againsignificantly greater than the low dose OP-1 sites (2.0/4.0, at α=0.05).No significant differences were found for implant type when comparingmean grades for cortex development, residual implant, and inflammatoryresponse.

Low Dose OP-1 Sites without CMC

New bone formation was apparent in all defects treated with low doseOP-1, but the amount of new bone within the defect often did not fillthe defect and was not continuous with the host bone ends. In one sitethe defect completely united histologically. New bone was in the earlystages of organization and remodeling. Some areas of newly mineralizingbone were also evident.

Low Dose OP-1/CMC Sites

The low dose OP-1/CMC sites had a similar histologic appearance comparedto the low dose OP-1 sites. However, and unexpectedly, new bone wascontinuous with the host bone more frequently in the low dose OP-1/CMCsites compared to the low dose OP-1 sites. In cases where the bone wascontinuous with the host bone, early remodeling and densification of thenew bone borders was apparent. In cases where new bone healing was notcomplete, areas of newly mineralizing bone were apparent, as well asareas of fibrous tissues within the defect. In general, the new bone waswell contained. Some areas of advanced remodeling along the defectborders was observed.

Standard OP-1 Device Sites

Extensive new bone formation bridged all defects. Early densification ofthe new bone borders had occurred. In some cases, areas of newlymineralizing bone joined areas of mature bone. At the center of thedefects, occasional small areas of residual carrier material waspresent. No inflammatory response was observed. New bone often extendedinto the soft tissues. Remodeling was most advanced at the defect/newbone borders. The bone had remodeled to a lamellar structure in theseareas.

Standard Dose OP-1/CMC Sites

There were no marked differences in the histologic appearance betweenthe standard OP-1 sites and the standard dose OP-1/CMC sites. Extensivenew bone spanned and filled the defects. The most extensive remodelingoccurred at the new bone/host bone borders. The remodeled bone had alamellar structure in these areas. Densification of new cortices wasevident, but not yet complete. Occasional small amounts of trappedresidual carrier material surrounded by new bone formation wereobserved. There was no associated, inflammatory response.

Conclusion

Improved osteogenic devices unexpectedly induced earlier and largervolumes of new bone formation at low doses of OP-1 than were induced bystandard devices at low doses of OP-1. Moreover, and unexpectedly,defect sites treated with improved osteogenic devices achieved thegreatest mean torsional load to failure at twelve-weeks post-operative.Histologically, improved devices unexpectedly achieved the greatest meanscore and more frequently demonstrated continuous new bone with hostbone.

B. Repair of Non-Critical Size Segmental Defects Using ImprovedOsteogenic Devices Containing Carboxymethylcellulose

1. Experiment 1: Time Course of Repair of Closed Defect as Treated Witha Unitary Device (Dogs)

This non-critical size gap study was conducted to evaluate injectableconfigurations of improved osteogenic devices. The study design used the3 mm gap at 4 week model. The study evaluated the healing of the defectafter injection of OP-1/CMC/collagen matrix configuration. Thecontralateral arm of each animal was a control. In addition, a healingtime course for an untreated defect was evaluated at 4, 8 and 12 weeks.

The details of the protocol used are summarized below.

Test System

Adult mongrel dogs (18) bred for purpose were utilized in this studybecause of their anatomical size and known bone repair and remodelingcharacteristics. The animals were approximately 2 to 4 years old atonset of study and weighed 20 to 30 kg (approximately). The animals wereradiographically screened to ensure proper size, skeletal maturity, andthat no obvious osseous abnormalities exist.

Test Material Description

Improved osteogenic device formulations comprising recombinant humanosteogenic protein-1 (rhOP-1) in a collagen matrix admixed with CMC weretested. Controls consisted of mock device alone.

-   -   Formulation 1: 0.350 mg rhOP-1 in 100 μl CMC gel (7%) w/o        collagen matrix    -   Formulation 2: 0.350 mg rhOP-1 in 100 μl acetate/lactose buffer    -   Formulation 3: 0.350 mg rhOP-1 in 170 mg collagen-CMC matrix        wetted with saline    -   Control 1: 0 mg rhOP-1 in 100 μl gel    -   Control 2: 0 mg rhOP-1 in 100 μl acetate/lactose buffer    -   Control 3: 0 mg rhOP-1 in 170 mg collagen-CMC matrix wetted with        saline

Experimental Design

Bilateral 3 mm ulna segmental defects were created in all animals. Nineanimals received one of the three experimental test formulations in theright side defect, such that three sites of each type were studied. Theleft defect was implanted with mock device. These animals weresacrificed at four weeks post-operative. The remaining nine animalsreceived non-implanted defects bilaterally and were sacrificed atperiods at four, eight, and 12 weeks (three at each time period). Asdiscussed above, radiographs were taken to study the progression ofhealing. Final determination of sacrifice dates of the nine animalsreceiving rhOP-1 formulations was based upon the weekly radiographs. Atsacrifice, all ulnae were retrieved en bloc and mechanically tested intorsion. Segments were evaluated by histology, as described above, fortissue response, and quality and amount of new bone formation, andextent of healing.

Using standard surgical techniques, a lateral incision approximately twocentimeters in length was made, and exposure of the ulna was obtainedusing blunt and sharp dissection. The 3 mm defect was created in theright mid-ulna using an oscillating saw. The radius was maintained formechanical stability, but no internal or external fixation was used. Thesoft-tissues were meticulously closed in layers around the defect. TherhOP-1 sample or mock device was then injected into the site as per thetreatment schedule. The procedure was then repeated on the contralateralside with the appropriate sample.

Radiographs of the forelimbs were obtained weekly until six weekspost-operative and then biweekly until 12 weeks in the survivinganimals. One additional x-ray was obtained from the remaining animals atsacrifice at twelve weeks post-operative. Radiographs were graded by theinvestigator on a 0-6 grading scale and compared to earlier radiographsto appreciate quality and speed of defeat healing.

Testing Procedures

As discussed above, the animals were sacrificed at the designated times,and the ulna and radius were immediately harvested en bloc. Both ulnawere macrophotographed and contact radiographs taken. Soft tissues weremeticulously dissected away from the defect site. A water-cooled saw wasused to cut the ulna to a uniform length of 9 cm with the defect sitecentered in the middle of the test specimen. Immediately aftersectioning, the specimen was tested in torsion to failure on an MTSclosed-loop hydraulic test machine (Minneapolis, Minn.), as describedabove.

Both tested and untested specimens were prepared for histologicevaluation, as already described above. Following microradiography, thesections were further ground to approximately 50 μm and stained withbasic fuchsin and toluidine blue for histologic evaluation of parametersof repair including: the quality of the union, the appearance andquality of the cortical and cancellous bone, and the inflammatoryresponse.

Descriptive statistics of mechanical testing, radiographic grading andhistology were evaluated to characterize healing.

Results

The following observations and representative data were collected todate (4 weeks post-operative):

Mechanical testing summaries appear in Tables 9, 10 and 11. Table 11 isa summary of control subjects in previous, unrelated experiments.Generally and overall, the results of this study indicate that animalstreated with OP-1 exhibit accelerated healing. The OP-1 treated defectshealed in one-third to one-half the time of untreated controls.Additionally, and unexpectedly, the CMC/OP-1/collagen formation resultedin better bone containment than observed in the absence of CMC. Theseobservations were confirmed mechanically, radiographically andhistologically.

Conclusion

CMC-containing osteogenic devices (injectable configuration) can be usedto repair non-critical size, 3 mm ulna segmental defects at a closeddefect site.

TABLE 9 Evaluation of Improved Device Formulations of rhOP-1 for Repairof Noncritical Size Defects MECHANICAL TESTING RESULTS Maximum LoadPercent intact Energy absorbed Animal to Failure Torque control tofailure Number Side Time Period Implant (N) (Nm) (%) Angulation(Nm-degrees) 18750 right 4 weeks FORMULATION 1 49.37 2.96 75.65 35.7263.57 18643 right 4 weeks FORMULATION 1 16.56 0.99 25.37 14.04 6.7818043 right 4 weeks FORMULATION 1 33.32 2.00 51.05 43.62 54.56 MEAN33.08 1.99 50.69 31.13 41.64 STANDARD 16.41 0.98 25.14 15.32 30.52DEVIATION SAMPLE SIZE 3 3 3 3 3  17884* right 4 weeks FORMULATION 232.47 1.95 49.75 50.11 55.91 18473 right 4 weeks FORMULATION 2 43.832.63 67.15 37.53 51.77 right 4 weeks FORMULATION 2 10.79 0.65 16.5320.78 5.94 MEAN 20.03 1.74 44.48 36.14 37.87 STANDARD 16.79 1.01 25.7214.71 27.73 DEVIATION SAMPLE SIZE 3 3 3 3 3 18772 right 4 weeksFORMULATION 3 42.64 2.56 65.33 55.06 55.74 18640 right 4 weeksFORMULATION 3 20.95 1.26 32.10 24.77 14.62 18508 right 4 weeksFORMULATION 3 7.24 0.43 11.09 17.08 3.04 MEAN 23.61 1.42 36.17 32.3024.47 STANDARD 17.85 1.07 27.35 20.08 27.70 DEVIATION SAMPLE SIZE 3 3 33 3 *Calculations based on raw data from mechanical testing printout.

TABLE 10 Evaluation of Improved Device Formulations of rhOP-1 for Repairof Noncritical Size Defects MECHANICAL TESTING RESULTS Maximum LoadPercent intact Energy absorbed Animal to Failure Torque control tofailure Number Side Time Period Implant (N) (Nm) (%) Angulation(Nm-degrees) 18750 left 4 weeks CONTROL 1 12.81 0.77 19.63 33.26 14.0618643 left 4 weeks CONTROL 1 11.00 8.00 16.85 59.35 16.83 18043 left 4weeks CONTROL 1 4.14 0.25 6.34 7.46 0.70 MEAN 9.32 3.01 14.27 33.3610.53 STANDARD 4.57 4.33 7.01 25.95 8.62 DEVIATION SAMPLE SIZE 3 3 3 3 3 17884* left 4 weeks CONTROL 2 4.82 0.29 7.38 11.12 0.73 18473 left 4weeks CONTROL 2 4.53 0.27 6.94 30.63 2.50 left 4 weeks CONTROL 2 7.520.45 11.52 33.29 8.59 MEAN 5.62 0.34 8.62 24.91 3.94 STANDARD 1.65 0.102.53 12.03 4.12 DEVIATION SAMPLE SIZE 3 3 3 3 3 18772 left 4 weeksCONTROL 3 15.41 0.92 23.61 49.91 15.40 18640 left 4 weeks CONTROL 310.28 0.62 15.75 40.50 11.23 18508 left 4 weeks CONTROL 3 4.32 0.26 6.624.89 0.41 MEAN 10.00 0.60 15.33 31.77 9.01 STANDARD 5.55 0.33 8.50 23.757.74 DEVIATION SAMPLE SIZE 3 3 3 3 3 *Calculations based on raw datafrom mechanical testing printout.

TABLE 11 Evaluation of Improved Device Formulations of rhOP-1 for Repairof Noncritical Size Defects - Unrelated Controls MECHANICAL TESTINGRESULTS Maximum Load Percent Energy absorbed Animal Time to FailureTorque intact control to failure Number Side Period Implant (N) (Nm) (%)Angulation (Nm-degrees) 17932 right 8 weeks Unrelated Control 35.40 2.1254.24 61.58 34.63 18926 right 8 weeks Unrelated Control 6.20 0.37 9.5059.65 6.96 18754 right 8 weeks Unrelated Control 25.68 1.54 39.34 33.5327.87 17932 left 8 weeks Unrelated Control 29.14 1.54 39.34 33.53 27.8718926 left 8 weeks Unrelated Control 5.67 0.34 8.69 47.20 6.20 18754left 8 weeks Unrelated Control 12.23 0.73 18.74 39.43 15.10 MEAN 19.051.14 29.19 44.64 18.43 STANDARD 12.68 0.76 19.42 14.14 11.36 DEVIATIONSAMPLE SIZE 6 6 6 6 6

2. Experiment 2: Accelerated Repair of a Closed Fracture Defect asTreated With a Unitary Device (Dogs)

The following is a comparative experimental study of the efficacy ofinjectable, CMC-containing rhOP-1 formulations for accelerating fracturehealing in dogs.

Test System

Adult male mongrel dogs bred for purpose were utilized in this study.Special attention was paid in selecting animals of uniform size andweight to limit the variability in bone geometry and loading. Theanimals were screened clinically and radiographically to exclude acuteand chronic medical conditions during a two-week quarantine period.

Using standard aseptic techniques, surgery was performed underisofluorane gas anesthesia and was monitored by electrocardiogram andheart rate monitors. Pre-surgical medication was administeredapproximately 20-30 minutes prior to anesthesia induction. Thepre-surgical medication consisted of atropine (dosage 0.02 mg/lb bodyweight) and acepromizine (dosage 0.1 mg/lb body weight). Anesthesia wasadministered by intravenous injection of sodium pentothal at the dosageof 5.0 mg/lb body weight. Following induction, an endotracheal tube wasplaced and anesthesia was maintained by isofluorane inhalation. Bothforelimbs were prepped and draped in sterile fashion. A lateral incisionapproximately two centimeters in length was made and exposure of theulna was obtained using blunt and sharp dissection. The 3.0 mmnoncritical sized defect was created in the mid-ulna using anoscillating saw. The radius was maintained for mechanical stability andno internal or external fixation was used. The site was irrigated withsaline and the soft tissues meticulously closed in layers around thedefect. The appropriate implant device was injected into the defect siteas per the treatment schedule. The procedure was then repeated on thecontralateral side with the appropriate implant.

Acepromizine (0.75 cc/501b body weight) and butorphanol tartrate (0.025mg/lb body weight) was administered as required postoperatively. Animalswere administered intramuscular antibiotics for four days post-surgeryand routine anterior-posterior radiographs was taken immediately aftersurgery to insure proper surgical placement. Animals were kept in 3×4foot recovery cages until weight bearing was demonstrated after whichthey were transferred to runs and allowed unrestricted motion.

Radiographs of the forelimbs were obtained weekly until four weeks, andthen biweekly to 16 weeks in surviving animals using standardizedexposure times and intensities. Radiographs were evaluated and comparedto earlier radiographs to appreciate quality and speed of defecthealing. Changes in radiographic appearance were evaluated based onpresence and density of new bone formation, extent of defect bridgingand incorporation of the host bone cortices.

Test Material Description

The implant materials consisted of recombinant human osteogenicprotein-1 (rhOP-1) in an acetate buffer formulation and rhOP-1 inCMC-collagen. The rhOP-1 formulations were compared to vehicle onlycontrols. The acetate buffer rhOP-1 formulation consisted of 3.5 mg/mlOP-1 in a lactose/acetate buffer delivered in a 100 μl volume. Thevehicle control consisted of a 100 μl volume of lactose/acetate buffer.The rhOP-1/CMC-collagen formulation consisted of 0.35 mg rhOP-1 in 170mg CMC-collagen matrix wetted with approximately 0.43 ml of saline andhad the consistency of a paste. The control CMC-collagen consisted of170 mg CMC-collagen matrix wetted with approximately 0.43 ml of salineand was also delivered in a 100 μl injectable volume.

Experimental Design

A total of 36 adult mongrel dogs were utilized. Bilateral ulna segmentaldefects, 3.0 mm in length, were created in all animals. Fourteen animalsreceived an injection of 0.35 mg rhOP-1/acetate buffer formulation inone defect and the acetate buffer without rhOP-1 in the contralateraldefect. Nine animals received an injection of 03.5 mgrhOP-1/CMC-collagen formation in one defect and CMC-collagen alone inthe contralateral defect. the 23 animals were sacrificed at periods of4, 8 and 12 weeks postoperative. Thirteen dogs received bilateraldefects with no implant (defect only) and were evaluated at periods of4, 8, 12 and 16 weeks postoperative.

Testing Procedures

At the end of the study period, animals were sacrificed using anintravenous barbiturate overdose. The ulna and radius were immediatelyharvested en bloc and placed in saline soaked diapers. Both ulna weremacrophotographed and contact radiographs taken before soft tissues werecarefully dissected away from the defect site. A watercooled saw wasthen used to cut the ulna to a uniform length of 9 cm with the defectcentered in the middle of the test specimen for biomechanical testingevaluation.

If defect healing was sufficient based upon manual manipulation,specimens were tested to failure in torsion on an MTS closed-loophydraulic test machine (Minneapolis, Minn.) operated in stroke controlat a constant displacement rate of 50 mm/min. Each end of the bonesegment was mounted in a cylindrical aluminum sleeve and cemented withmethylmethacrylate. One end was rigidly fixed and the other was rotatedcounterclockwise. Since the dog ulna has a slight curvature, thespecimens were mounted eccentrically to keep specimen rotation coaxialwith that of the testing device. The torsional force was applied with alever arm of 6 cm. Force-angular displacement curves were generated fromwhich the torque and angular deformation to failure were obtained, andthe energy absorption to failure computed as the area until theload-displacement curve.

Both tested and untested specimens were prepared for histologicevaluation. The individual specimens were fixed by immersion in 10%buffered formalin solution immediately following mechanical testing orafter sectioning in untested specimens. On a water cooled diamond sawthe specimens were divided by bisecting the specimen down its long axis.This procedure resulted in two portions of each specimen for histologicpreparations including undecalcified ground sectioning and undecalcifiedmicrotome sectioning. The histologic sections were evaluated for thequality of union, the appearance and quality of the cortical andcancellous bone, and bone remodeling.

Results Gross Observations

All rhOP-1 treated defects had new bone formation as early as 4 weekspostoperative. All treated defects were manually stable and bridged withsolid new bone that began to remodel between 8 and 12 weekspostoperative. In some defects, the new bone extended beyond the defectends and into the overlying soft tissues surrounding the defects.

Most control defects were not completely stable upon manual manipulationat 4 weeks postoperative, although most were mechanically tested.Fibrous tissue was often present and defect ends remained visible withsome signs of new bone. By 12 weeks postoperative, most control defectswere stable with only occasional slight motion of the defect ends.

Radiographic Evaluation

In the rhOP-1 treated defects, traces of new bone were seen by two weekspostoperative in and around the defect sites. The amount and density ofnew bone increased from 2 to 4 weeks with the host bone corticesbeginning the obscure. Between 4 and 8 weeks postoperative, rhOP-1treated defects had significant amounts of radiodense new bone at thedefect ends and bridging the defect laterally. By 12 weeks, the hostcortices were obscured with radiodense bridging bond.

The radiographic appearance of the treated and untreated control defectswas significantly different from the appearance of the rhOP-1 treateddefects. Between 2 and 3 weeks postoperative, there were no significantchanges in the radiographic appearances compared to postoperativeappearances. By 4 weeks, faint changes in the radiodensity of the hostbone defect ends were visible. From 8 to 12 weeks, some new boneextended from the endosteal regions and host bone ends, althoughbridging was not complete. By 16 weeks postoperative, only one-half ofthe untreated controls showed radiographic signs of complete bony defecthealing.

Mechanical Testing

The mean mechanical testing results by treatment group and time periodare summarized in Tables 11A and 11B. Torsional strengths of defectstreated with rhOP-1 were significantly greater than untreated controlsand vehicle only controls and approached the strength of previouslytested intact ulnae. The mechanical strength of the rhOP-1/acetatebuffer formulation defects was 59% of intact ulna strength at 4 weekspostoperative, 77% at 8 weeks postoperative, and 98% at 12 weekspostoperative. The mechanical strength of the rhOP-1/CMC-collagendefects was 36%, 53% and 66% of intact strength at 4 weeks, 8 weeks and12 weeks, respectively.

Mechanically, the control defect sites had little mechanical stabilityat the early time periods although defect strength did improve withtime. The mechanical strength of defects receiving the control acetatebuffer solution was between 23% to 30% of the rhOP-1/acetate buffertreated defects at equivalent time periods. The control defects had amechanical strength equivalent to 16% of intact ulna strength at 4 weekspostoperative, 18% at 8 weeks, and 29% at 12 weeks. The CMC-collagenonly defects were similar in mechanical strength to the control acetatebuffer defects. The mechanical strength of untreated defects increasedfrom 9% at 4 weeks to 70% at 12 weeks postoperative. The mean mechanicalstrength at 16 weeks postoperative decreased to 28% which was similar tothe 8-week strength of 29%.

TABLE 11A Mechanical testing results, mean ± standard deviation (n)Maximum Energy absorbed Load to Torque Percent Intact Angulation toFailure Implant Weeks Failure (N) (Nm) Control (%) (degrees)(Nm-degrees) rhOP-1/ 4 weeks 38.46 ± 17.3 2.31 ± 1.0 58.92 ± 26.5 25.18± 16.2 35.71 ± 39.3 acetate buffer (8) (8) (8) (8) (8) rhOP-1/ 8 weeks50.57 ± 23.0 3.03 ± 1.4 77.48 ± 35.3 39.56 ± 14.6 70.51 ± 38.0 acetatebuffer (3) (3) (3) (3) (3) rhOP-1/ 12 weeks  63.70 ± 22.1 3.82 ± 1.397.60 ± 33.8 18.79 ± 2.4  40.40 ± 7.2  acetate buffer (3) (3) (3) (3)(3) Acetate buffer 4 weeks 10.72 ± 6.4  0.64 ± 0.4 16.42 ± 9.7  23.85 ±22.3 5.46 ± 7.9 only (8) (8) (8) (8) (8) Acetate buffer 8 weeks 11.47 ±9.4  0.69 ± 0.6 17.57 ± 14.5 39.38 ± 32.4 11.83 ± 10.2 only (3) (3) (3)(3) (3) Acetate buffer 12 weeks  18.91 ± 29.9 1.13 ± 1.8 28.97 ± 45.935.72 ± 11.4 43.69 ± 72.7 only (3) (3) (3) (3) (3) rhOP-1/ 4 weeks 23.61± 17.9 1.42 ± 1.1 36.17 ± 27.4 32.30 ± 20.1 24.47 ± 27.7 CMC-collagen(3) (3) (3) (3) (3) rhOP-1/ 8 weeks 34.33 ± 22.6 2.06 ± 1.4 52.60 ± 34.642.76 ± 20.2 37.08 ± 14.4 CMC-collagen (3) (3) (3) (3) (3) rhOP-1/ 12weeks  43.39 ± 22.3 2.60 ± 1.3 66.47 ± 34.2 32.07 ± 10.1 47.65 ± 21.8CMC-collagen (3) (3) (3) (3) (3) CMC-collagen 4 weeks 10.00 ± 5.55 0.60± 0.3 15.33 ± 8.5  31.77 ± 23.8 9.01 ± 7.7 only (3) (3) (3) (3) (3)CMC-collagen 8 weeks 4.45 ± 4.0 0.27 ± 0.2 6.81 ± 6.2 31.90 ± 25.6 5.09± 6.3 only (3) (3) (3) (3) (3) CMC-collagen 12 weeks  18.82 ± 8.6  1.13± 0.5 28.84 ± 13.2 43.87 ± 11.0 23.55 ± 16.4 only (3) (3) (3) (3) (3)Untreated 4 weeks 6.04 ± 1.8 0.36 ± 0.1 9.25 ± 2.8 43.71 ± 12.3 6.00 ±1.8 (5/8 tested) (5) (5) (5) (5) (5) Untreated 8 weeks 19.05 ± 12.7 1.14± 0.8 29.19 ± 19.4 44.64 ± 14.1 18.43 ± 11.4 (6) (6) (6) (6) (6)Untreated 12 weeks  45.91 ± 40.6 2.75 ± 2.4 70.34 ± 62.1 38.04 ± 17.838.26 ± 21.4 (6) (6) (6) (6) (6) Untreated 16 weeks  18.55 ± 9.3   1.11± 0.56 28.43 ± 14.2 39.15 ± 8.3  20.19 ± 9.8  (5/6 tested) (5) (5) (5)(5) (5)

TABLE 11B Mechanical testing results in terms of percentage of intactulna strength, mean ± standard deviation (n) Implant 4 weeks 8 weeks 12weeks 16 weeks rhOP-1 59 ± 26 77 ± 35 98 ± 34 — acetate buffer (8) (3)(3) Acetate buffer 16 ± 10 18 ± 15 29 ± 46 — only (8) (3) (3) rhOP-1 36± 27 53 ± 35 66 ± 34 — CMC-collagen (3) (3) (3) CMC-collagen 15 ± 9  7 ±6 29 ± 13 — only (3) (3) (3) Untreated 9 ± 3 29 ± 19 70 ± 62 28 ± 14 (5)(6) (6) (5)

Histologic Evaluation

The histology of the rhOP-1 and control defects correlated well withgross, radiographic and mechanical testing results. In the rhOP-1treated defects, proliferative new bone formation was observed spanningthe defects and in some cases extending into the subcutaneous tissue.New bone formed from the endostreal ulna regions and from the periosteumnear the defect cortices. Bridging with new bone was generally completedby 8 weeks postoperative, although areas of mineralizing cartilage werepresent. Defects were bridged and filled with dense woven bone andreorganization of the host bone cortices was observed by 12 weeks.

Treated and untreated control defects showed only signs of fibroustissue union with small amounts of new bone formed along the lateralulna periosteum or from the endosteal region at 4 weeks postoperative.At 8 weeks, fibrocartilage filled the control defects with areas ofmineralizing cartilage present between new bone growth. Significantamounts of new bone formed at the host bone cortices and extended intothe defects. Defect cortices were obscured with dense new bone formationand endochondral healing was advanced, although union was not complete.By 16 weeks, control defects were bridged with new bone with some gapsof mineralizing cartilage present. New bone extended from the hostcortices and adjacent periosteal tissue layers across the defect.

Conclusion

The results of this study demonstrate that osteogenic proteins injectedinto noncritical sized defects can accelerate bone repair. The localpercutaneous injection of rhOP-1 to the noncritical sized defect in thecanine ulna resulted in a proliferative periosteal and endosteal newbone formation compared to untreated and vehicle only treated controldefects. Radiographically, the rhOP-1 injection resulted in diffusecalcifications of new bone and early fracture callus formation as earlyas 2 to 3 weeks postoperative with significant bone bridging andincorporation of the host cortices by 8 to 12 weeks postoperative. Themechanical strength of noncritical sized defects treated with rhOP-1approached the strength of intact ulna at 12 weeks and were 2 to 3 timesthat observed in control defect healing.

C. Repair of Fracture Defects Using Improved Osteogenic DevicesContaining Carboxymethylcellulose 1. Experiment 1: Goat Fracture StudyUsing Varying Doses of OP-1 (Closed Defect Site)

The following is a comparative randomized experimental study of freshclosed tibial midshaft fracture defects (distracted to 5 mm) in goats.

Choice of Experimental Animal

It is generally recognized in the art that goats have a bone healingrate comparable to that of humans. Thus, the results of this study canbe extrapolated to a clinical setting.

Moreover, it is appreciated by the skilled artisan that the bones ofgoats show similarity to those of humans regarding size, shape andmechanical load.

As disclosed and described herein, an animal model for a closeddiaphyseal fracture has been developed. This model promotes the study ofnatural and accelerated fracture healing, with or without an internalfracture fixation device, by permitting creation of a reproduciblestandard fracture of the hind limb. Briefly, in fully anesthetizedgoats, a closed fracture of the midshaft of the tibia is created withthe aid of a three point bending device. After closed reduction anddistraction to 5 mm of the fracture, an external cast is applied.Because of a decrease in the swelling of the hind limb, the cast isreplaced biweekly to retain stability. After 2 weeks, the animals arefull weight bearing on the fractured limb, and after 4-6 weeks thefracture is healed clinically and radiographically. The cast is removedafter 6 weeks.

The animals are purchased from Ruiter (Netherlands), a goat breedingspecialist. Random bred adult female milk-goats will be used. Tocircumvent the influence of a developing skeleton on the results, adultanimals will be used. The animals are skeletally mature, 1 to 2 yearsold and weigh about 50 kg.

Experimental Procedure

As a premedication, ketamin 10 mg/kg i.m. and atropine 1.5 mg i.m. (orart-recognized equivalents of the foregoing medications) areadministered about 15 minutes before fully anesthetizing the animals.The latter is accomplished with etomidates (or art-recognizedequivalents thereof) 0.3 mg/kg i.v. After intubation, anesthesia ismaintained with an O₂/N₂O-mixture (1:1, vol/vol) supplemented with 1 to2% isoflurane (or art-recognized equivalents thereof).

With a 3-point bending device, a varus trauma is applied to the lefttibia until a closed midshaft fracture is obtained. The fracture is thenreduced manually, and the skin over the fracture area is shaved. Thewhole left hind limb is iodinated with an alcohol containingdisinfectant solution for closed osteogenic device administration byinjection and to dry the skin for subsequent cast immobilization. Theosteogenic device is injected at the fracture site in the vicinity ofthe fracture gap to maximize contact with the medullary cavity. Forexample, an osteogenic device is injected intramedullary with a thickbone marrow aspiration needle. After the injection, cast immobilizationis applied.

Study Design

The animals are divided into 5 groups (I-V) of 3 animals and 1 group(VI) of 9 animals according to treatment: 0.5 mg OP-1 in an injectableconfiguration of osteogenic device containing at least OP-1, collagenmatrix, and binding agent such as CMC, formulated as described above(directly after creation of the fracture) (Group I), 1.0 mg OP-1 in aninjectable device containing at least OP-1, collagen, and binding agent,such as CMC, (directly after creation of the fracture) (Group IV), 1.0mg OP-1 in a standard configuration of OP-1 device (corresponding to 0.4grain OP-1 device) injected directly after creation of the fracture(Group V), and no treatment with OP-1 (Group VI, controls). Thetreatment groups are summarized as follows:

Time of Approximate injection Amount of Number of Group (days) DeviceOP-1 (mg) animals I 0 Injectable 0.5 3 II 0 Injectable 1.0 3 III 3Injectable 1.0 3 IV 0 Injectable 1.0 3 V 0 Standard Device 1.0 3 VI NoneNone 0 9

The animals are sacrificed 2, 4 and 6 weeks after creation of thefracture. In groups I to V, one animal is sacrificed at each timeinterval, and in group VI, three animals are sacrificed at each timeinterval. By comparing the treated groups to the controls, theaccelerating effect of treatment on fracture healing can be determined.Information about the OP-1 dose effect and the time of injection can beobtained by comparison of group I to group II, respectively, and groupII to group III. Differences in efficacy between differentconfigurations are assessed by evaluating the results of groups II, IVand V.

In other related experiments, doses of osteogenic protein such as OP-1will range from approximately 0.125 to 10.0 mg. Certain otherconfigurations of improved osteogenic devices will contain varyingamounts of binding agent such as CMC, ranging from below 200 mg CMC/1000mg collagen matrix to above 200 mg CMC/1000 mg collagen matrix. Wettingagent volumes will be varied as earlier described to achieve the desiredconsistency/configuration of osteogenic device. In yet other relatedexperiments, other binding agents such as fibrin glue and/or othermatrices such as β-TCP will be used.

Evaluating Defect Repair Radiography

X-rays are made following a standardized procedure and depict thefracture site in two directions, anteroposterior and mediolateral. Thefirst radiographs are taken immediately after creation of the fractureand thereafter biweekly until sacrifice of the animals. The radiographsat the time of sacrifice are made after removal of the casting material;all others are made with the casting material in situ. They are judgedqualitatively by two blinded radiologists or surgeons, and, if possible,the following grading scale for evaluating the healing process isapplied:

Grade 0: No difference compared to directly after creation of thefracture

Grade, 1: Small amount of callus

Grade 2: Moderate amount of callus

Grade 3: Large amount of callus

Grade 4: Fading of the fracture ends

Special attention is paid to the type of fracture and alignment.

Computed Tomography

After removal of the left hindlimb and casting material, and aftermaking of the radiographs, a CT-scan of the fracture area is made. Thesoft tissues should remain in situ for a better quality of scans.Remnants of the fracture gap and callus can be made visible in this way.Moreover, the amount of callus can be calculated. More detailedinformation about the progress of the healing process can be obtainedwith CT scans than with plain radiographs.

Biomechanical Test

After CT scanning and subsequent removal of all soft tissues from thetibia, biomechanical investigations are performed. A method for advancedmechanical testing of bone is developed as follows: the bendingstiffness in 24 directions at angular increments of 15° is measured anddepicted as a vector in a X-Y coordinates system, by which an ellipse isobtained. The ellipse is compared with that of the contralateral intacttibia. Parameters can be derived from this comparison that serve asmeasures of the healing efficiency. Finally, a torsion-test-to-failureis done and the measured torsion strength, torsion stiffness, angulardisplacement and energy absorption-to-failure is expressed as apercentage of the contralateral healthy tibia. This comparison with thecontralateral tibia is made to reduce the interindividual variation.

Histology

After biomechanical testing, the bone fragments are held together withspecial rings for histologic examination. Standard fixation, imbeddingand staining techniques for bone and cartilage are used. Specialattention is paid to signs of fibrous, osteochondral or bony union. Ahistologic scoring system is applied to quantitate the amount of fibroustissue, cartilage, newly formed bone and bone marrow in the fracturegap.

Experimental Results

It is expected that mechanical, radiographic, tomographic andhistological data will indicate that injectable configurations ofimproved osteogenic devices can induce accelerated repair of closed sitefracture defects.

Conclusion

Improved osteogenic devices (injectable configuration) can be used torepair fresh tibial midshaft fracture defects (distracted to 5 mm) at aclosed defect site.

2. Experiment 2: Goat Fracture Study Using Varying Doses of OP-1 atVarying Times (Closed Defect Site)

This independent study also uses goats as the animal model for studyingrepair of fracture defects using improved osteogenic devices. Usingtechniques similar to those described above, fresh closed diaphysealfractures (mostly transverse and simple oblique) with reduction withexternal fixation and distraction to 5 mm are treated usingCMC-containing osteogenic devices.

The study design is as follows:

Group Treatment No. Goats I No injection 10 II CMC + collagen alone viainjection 10 III CMC + collagen + OP-1 (2.5 mg 10 OP-1/1000 mg collagen)via injection IV CMC + collagen + OP-1 (half-maximal 10 dosage of 1.25mg OP-1/1000 mg collagen) via injectionFive goats in each group are sacrificed at 2 weeks post-treatment andfive goats in each group are sacrificed at 4 weeks post-treatment.

Other related studies investigate repair of fracture defects at timepoints greater than 4 weeks, and investigate both lower and higherdosages of OP-1. Additionally, repair of fracture defects usingdiffering total amounts (mg) of the CMC-containing OP-1 deviceadministered at the defect site are studied. One study utilizes a deviceof 400 mg total weight administered at the defect site. Yet otherrelated studies will utilize any if the aforementioned binding agents,such as fibrin glue, and/or any of the aforementioned matrices, such asβ-TCP.

Defect repair is evaluated using a variety of routine clinicalprotocols, including radiography, CT scan, biomechanical testing, andhistology, as described in more detail above.

Experimental Results

It is expected that mechanical, radiographic, tomographical,histological data will indicate that injectable configurations ofimproved osteogenic devices can induce accelerated repair of closed sitefracture defects. It is also anticipated that, in certain preferredembodiments, low doses of osteogenic protein will be effective to inducerepair, especially in improved osteogenic devices.

Conclusion

Improved osteogenic devices (injectable configuration) can be used torepair fresh closed diaphyseal fractures (distracted to 5 mm) at aclosed defect site.

D. Repair of Osteochondral Defects Using Improving Osteogenic DevicesContaining Carboxymethylcellulose

1. Experiment 1: Full-Thickness Osteochondral Defects (Dogs)

A study using the dog osteochondral plug defect model was conducted todemonstrate the efficacy of improved osteogenic devices for repairingosteochondral/chondral defects. Four formulations of implants wereevaluated, including, (1) standard osteogenic device, including rhOP-1and collagen matrix, (2) improved osteogenic device, including rhOP-1,collagen matrix and carboxymethylcellulose (CMC) binding agent, (3)collagen matrix only, or (4) collagen matrix and CMC binding agent.

Briefly, full thickness defects 5 mm in diameter and extending 6 mm intothe subchondral bone were created bilaterally on the medial femoralcondyle of 4 adult mongrel dogs. Adult male mongrel dogs were chosenbecause of their anatomical size and bone repair and remodelingcharacteristics. Special attention was paid in selecting animals ofuniform size and weight to limit the variability in bone geometry andjoint loading. The animals were radiographically screenedpre-operatively to ensure proper size, skeletal maturity, and that noobvious osseous abnormalities existed. The left side defects receivedstandard osteogenic device in two animals, and the improved osteogenicdevice in the other two animals. The right side defects received matrixalone in one animal, a matrix/binding agent mixture in one animal, andwas untreated in the remaining two animals.

Test Device Description

The standard osteogenic device consisted of rhOP-1 admixed with bovineType I bone collagen matrix (2.5 mg rhOP-1/g matrix). The improvedosteogenic device comprised 100 mg of the OP-1/collagen matrix standardosteogenic device combined with 20 mg of CMC (total of 120 mg). Controlsconsisted of bovine Type I bone collagen matrix alone, and the collagenmatrix with CMC. Both were supplied in 100 mg quantities.

Study Design

Study design is summarized in Table 12.

TABLE 12 Dog Osteochondral Defect Repair using OP-1 Animal Left RightNumber Implant Implant H122 OP-1 Matrix H130 OP-1 None H125 OP-1/CMCNone H132 OP-1/CMC None OP-1: 100 mg OP-1/Collagen Device (standardosteogenic device). OP-1/CMC: 120 mg OP-1/CMC/Collagen Device (improvedosteogenic device). Matrix: 100 mg Collagen. CMC/Matrix: 100 mgCMC/Collagen. Devices and controls were wetted with saline (approx. 0.21to 0.26 ml) to achieve a putty consistency prior to implantation.

Surgery

Using standard aseptic techniques, surgery was performed underisofluorane gas anesthesia. Anesthesia was administered by intravenousinjection of sodium pentothal at a dosage of 5.0 mg/lb body weight. Amedial parapatellar incision approximately four centimeters in lengthwas made. The patella was retracted laterally to expose the femoralcondyle. A 5 mm drill bit with a specially designed sleeve to preventover drilling of the defect depth (6 mm) was used to create the finaldefect. Sterile saline was added to the improved osteogenic device andmixed just prior to implantation. After irrigation of the defect withsaline to remove bone debris and spilled marrow cells, the appropriatedevice was packed into the defect site using a blunt probe. Enoughdevice was placed within the defect so that it was flush with thearticulating surface. The joint capsule and soft-tissues were thenclosed in layers. The procedure was repeated on the contralateral sidewith the appropriate implant.

Evaluations and Terminal Procedures

Osteochondral healing was evaluated grossly and histologically usingroutine protocols, as described below. Radiographs were utilized toevaluate healing.

At twelve weeks post-operative each animal was sacrificed by anintravenous barbiturate overdose. Both right and left distal femurs wereharvested en bloc and kept in cool saline until gross grading andmicrophotography were completed. The specimens were then placed in 4%paraformaldehyde fixative, labeled with all necessary identifications,and stored at 4° C. until shipped approximately 10 days post-sacrifice.Just prior to shipping the specimens were trimmed into small blocks,with the articular defect in the center.

Gross Analysis

Each harvested defect was graded for gross appearance. This analysisapportions points based upon the formation of intra-articular adhesions,restoration of articular surface, erosion and appearance of thecartilage. A total of eight points is possible. The gross grading scaleis set forth in Table 13.

TABLE 13 Gross Grading Scale Grades Intra-articular adhesions None = 2Minimal/fine loose fibrous tissue = 1 Major/dense fibrous tissue = 0Restoration of articular surface Complete = 2 Partial = 1 None = 0Erosion of cartilage None = 2 Defect site/site border = 1 Defect siteand adjacent normal cartilage = 0 Appearance of cartilage Translucent =2 Opaque = 1 Discolored or irregular = 0 TOTAL SCORE 8 possible points

Histology

All specimens were prepared for histologic evaluation. The individualspecimens were fixed by immersion in 4% paraformaldehyde solution. Inaddition, using routine procedures as described elsewhere herein, tissuetyping analysis was performed in order to characterize the collagen typeand percent tissue composition. Non-decalcified sections, one from eachspecimen, stained with Safranin-O and Fast Green stains (to indicateglycosaminoglycan content in the matrix), were returned for evaluation.

Histologic sections were based upon the nature of the repair cartilage,structural characteristics, and cellular changes. The histologic gradingscale is set forth in Table 14.

TABLE 14 Histology Grading Scale Nature of the Predominant Tissue:Cellular morphology Hyaline articular cartilage = 4 Incompletelydifferentiated = 2 Fibrous tissue or bone = 0 Safranin-O stainingNormal/near normal = 3 of the matrix Moderate = 2 Slight 1 None = 0STRUCTURAL CHARACTERISTICS: Surface regularity Smooth/intact = 3Superficial horizontal lamination = 2 Fissures, 25-100% of thickness = 0Severe disruption, fibrillation = 0 Structural integrity Normal = 2Slight disruption, including cysts = 1 Severe disintegration = 0Thickness 100% of normal cartilage thickness = 2 50-100% = 1 0-50% = 0Bonding to the Bonded at both ends of the defect = 2 adjacent cartilageBonded at one end or partially bonded 1 at both ends = 1 Not bonded = 0FREEDOM FROM CELLULAR CHANGES OF DEGENERATION: Hypocellularity None = 3Slight = 2 Moderate = 1 Severe = 0 Chondrocyte None = 2 clustering <25%of cells = 1 >25% of cells = 0 Freedom from Normal cellularity, noclusters, 3 degenerative normal staining = changes in Normalcellularity, mild clusters, 2 adjacent cartilage moderate staining =Mild or moderate hypocellularity, 1 slight staining = Severehypocellularity, poor or no 0 staining = TOTAL 24  possible points

Results

All surgeries were uneventful with no post-operative complications. Ingeneral, some medial knee swelling was observed on post-operative dayfour bilaterally in all four animals and subsided by post-operative dayten. No animal experienced any adverse reaction related to the implantedmaterials or experimental procedures.

Gross Evaluation

A summary of the mean gross evaluation grades appears in Table 15.

TABLE 15 Mean Gross Evaluation Grade ± standard deviation (n) StandardImproved Osteogenic Osteogenic Collagen Collagen No Device Device MatrixOnly Matrix/CMC Treatment Intra- 2.0 ± 0.0 2.0 ± 0.0 2.0 ± 0.0 2.0 ± 0.02.0 ± 0.0 Articular Restoration 1.5 ± 0.6 1.5 ± 0.6 0.0 ± 0.0 0.5 ± 0.62.0 ± 0.0 of Surface Erosion 1.8 ± 0.5 1.23 ± 0.5  1.5 ± 0.7 1.0 ± 1.41.8 ± 0.5 Appearance 0.8 ± 0.5 1.0 ± 0.8 0.0 ± 0.0 1.0 ± 0.0 1.5 ± 0.6Total 6.0 ± 1.4 5.8 ± 1.7 3.5 ± 0.7 4.5 ± 0.7 7.3 ± 0.5 (out of 8 (2)(2) (1) (1) (2) possible points)

Histological Evaluation

The non-treated defects and defects treated with the improved osteogenicdevice of OP-1, collagen matrix and CMC received the greatest meanhistologic grade, 15 and 16.5 out 24 possible points, respectively. Ineach of these groups, however, one specimen looked markedly better thanthe other. The collagen matrix only, collagen matrix with CMC, and thestandard osteogenic device treated sites however, scored slightly moreconsistently, and lower than, the sites treated with improved osteogenicdevice (n≦2). A summary of the mean histological grades appears in Table16.

TABLE 16 Mean Histologic Evaluation Grade ± standard deviation (n)Standard Improved Collagen Osteogenic Osteogenic Matrix Collagen DeviceDevice Only Matrix/CMC Non Implanted Nature of the 3.5 ± 0.7 4.5 ± 2.1  1.0   2.0 4.0 ± 2.8 Predominant (2) (2) (1) (1) (2) Tissue Structural1.5 ± 0.1 5.5 ± 0.6   7.0   6.0 5.0 ± 2.8 Characteristics (2) (2) (1)(1) (2) Freedom from 4.5 ± 0.7 6.5 ± 2.1   3.0   4.0 6.0 ± 1.4 Cellular(2) (2) (1) (1) (2) Changes of Dengeneration Total 12.5 ± 0.7  16.5 ±7.8   11.0  12.0 1.5 ± 7.1 (out of 24 (2) (2) (1) (1) (2) possiblepoints)

Unexpectedly, sites treated with the improved osteogenic device achievedthe highest mean scores for the nature of the new repair tissue, for thestructural characteristics of the repair, and for minimizing thedegeneration of the repair cartilage or the surrounding intactcartilage. The improved osteogenic device sites also received thehighest overall total score. These results were weighted by the score ofone animal, in which the cellular and tissue morphology was consistentwith articular cartilage. The repair cartilage was continuous with theintact cartilage and the thickness of the repair was the same as theintact cartilage. The subchondral bone layer was also completelyrestored. Healing was not as advanced in the other sites treated withthe OP-1/collagen matrix with or without CMC. Lower scores were theresult of incomplete differentiation of the repair tissue, incompletesubchondral bone restoration, and uneven thickness of the repair.Residual implant or carrier material was not observed in any section.

Comparisons within animals demonstrated that, in three animals, thedefects receiving devices containing OP-1, with or without CMC (all leftdefects), achieved histologic grades equal to or greater than thecontralateral defect receiving the control matrix or no treatment.

Unexpectedly, the OP-1 device without CMC induced bone and cartilageformation, but in a more disorganized fashion with considerable fibroustissue present. Untreated or carrier alone samples were filled byfibrous cartilage and dense connective tissue.

These data suggest that the unexpected superior repair achieved withimproved osteogenic device is associated with the differences in itsconsistency relative to that of the standard osteogenic device withoutbinding agent, which in turn affects the containment of the device perse at the defect site. Formulation adhesion and disintegrationproperties are expected to be critical in articular cartilage defectsgiven the dynamic nature of the joint.

Immunostaining of Type I and Type II Collagen and Polarized LightMicroscopy

This study also stained sections to compare collagen repair at defectsites treated with: no device, two types of matrix only compositions(matrix and matrix/binding agent), or both matrix compositions withOP-1.

In general, using the collagen Type I antibody, staining of the existingunderlying subchondral bone, as well as the newly regenerated bone, wasobserved. The newly regenerated bone differed slightly from the existingbone by the presence of regions of more disorganized matrix when viewedunder phase contrast microscopy. Using the Type II collagen antibody,the existing articular cartilage stained qualitatively as well as thereparative tissue in the defects, although staining of the new tissuewas less intense. In at least one defect treated with improvedosteogenic device, complete regeneration of the subchondral bone wasobserved with articular-like cartilage regenerated along the top. Thecellular matrix of this regenerated cartilage was not identical to theexisting articular cartilage, but a visible cellular matrix composed oflarge loose bundles could be seen under phase contrast.

Defects Treated with Improved Osteogenic Device.

In defects treated with improved osteogenic device, at least one animalevidenced repair of articular cartilage at a macroscopic level. Thesubchondral bone was regenerated and a new cartilage layer of nearnormal thickness was seen by histological staining with toluidine blueand Safarnin O. These layers and tissues stained appropriately, withType I antibody localized in the subchondral bone and Type II collagenlocalized in the new cartilage-like layer. There was also some evidenceof the regeneration of a zone of calcified cartilage and distincttidemark in the regenerated cartilage. However, some differences wereseen between the new and existing articular cartilage layer. The newcartilage had a higher density of chondrocytes and contained loose,disorganized bundles of fibers visible by phase contrast microscopy orwith polarized light. It should be noted that only a single time pointduring the repair process is represented here and that the results oflonger or shorter periods is unknown.

Defects Treated with Standard Osteogenic Device.

Defects treated with standard osteogenic devices showed approximately50% of the bone was regenerated in the defect site with in-growth ofarticular cartilage from the edges of the defect. There appeared to besome additional areas of articular cartilage formation next to the newlyregenerated bone, with the remainder of the defect filled withreparative tissue. The reparative tissue stained lightly with collagenType II, and not with Type I collagen, antibodies. More chondrocyteswere present with large loose bundles of matrix surrounding the cells.Treatment with the standard osteogenic device differed from treatment ofthe improved osteogenic device in that the subchondral bone failed toregenerate to its normal level, and dense disorganized fibrous tissueappeared above the new cartilage, which caused the top of the defect tobulge with an irregular surface. This fibrous tissue appeared to havemore fibroblast-like cells with fibrous bundles arranged parallel to thearticular surface.

Defects Treated with Matrix/Binding Agent.

A defect with only matrix/binding agent without OP-1 showed regenerationof about one-third of the removed subchondral bone, with the remainderfilled with a reparative tissue This regenerated tissue stained lightlywith Type I collagen antibodies, especially near the bottom of thedefect, and showed stronger staining with the Type II collagen antibody,with strongest staining near the surface. A dense disorganized visiblematrix is apparent in the top half of the reparative tissue, and a moreorganized horizontal pattern of fibers appears in the bottom half.Toluidine blue did not stain the reparative tissue, whereas Safranin Ostained the top and bottom half differentially. The half near thearticular surface stained lightly with Safranin O, and the bottomstained with Fast Green. A similar distinction was observed between thetwo halves of the reparative tissue when stained with Masson Trichrom.Although the reparative tissue did not look like articular cartilage,the region near the articular surface did appear to contain Type IIcollagen, an acidic matrix with perhaps some mucopolysaccharides. Thebottom half had more Type I collagen with less carbohydrate and may bemore connective tissue-like in nature.

The single defect treated with collagen matrix alone did not show anyregeneration of the subchondral bone. The reparative tissue that filledthe defect stained lightly with both collagen Type I and II antibodies.This tissue had an increased fibrous matrix with fibroblastic like cellsand appeared in some areas to be similar to fibrocartilage. This samplewas similar to the treatment with the CMC/collagen matrix alone, withslight localization of both Type I and II collagen in the reparativetissue. In addition, the defect site showed the same differentialstaining with Safranin O/Fast Green, with staining of the top half ofthe reparative tissue with Safranin O and the bottom with Fast Green.

Summary And Conclusion

Osteochondral defects treated with the improved osteogenic devicesunexpectedly demonstrated more advanced cartilage regeneration,chondrocyte and cartilage phenotype compared to defects treated with thestandard osteogenic device, collagen matrix alone, or collagen matrixadmixed with CMC, all of which demonstrated less organized repaircartilage and subchondral bone formation. Poor repair by treatment withthe collagen matrix or collagen matrix with CMC indicates that thepresence of a collagen scaffold alone is not sufficient to inducehealing and may actually deter the progression of healing andorganization of repair tissue.

Full-thickness osteochondral defects can be repaired usingCMC-containing osteogenic devices in accordance with the methods of theinstant invention. It is expected that full-thickness osteochondraldefects can also be repaired using improved osteogenic devicescontaining any of the aforementioned preferred binding agents such asfibrin glue and/or any of the aforementioned preferred matrices.

2. Experiment 2: Long Term Evaluation of Repair of Full-ThicknessOsteochondral Defects (Dogs)

This study was conducted to further evaluate repair ofosteochondral/chondral defects by improved osteogenic devices. To date,the study examined the effects of the improved osteogenic device at 6and 12 weeks and will continue to examine effects at 26 and 52 weeks.This provides long term repair stability data. The organization of newcartilage over time was followed to determine if it approximates normaltissue with respect to its structure and function. Two formulations ofdevices were evaluated in osteochondral/chondral defects including: 1)improved osteogenic device, or 2) mock devices containing CMC andcollagen matrix only.

Briefly, full thickness defects 5 mm in diameter extending 6 mm into thesubchondral bone were created bilaterally on the medial femoral condyleof 16 adult mongrel dogs. Adult mongrels were utilized in this studybecause of their anatomical size and known bone repair and remodelingcharacteristics. All animals were between 1 and 4 years old and weighapproximately 20 to 30 kg. Specific attention was paid to selectinganimals of uniform size and weight to limit the variability in jointloading. The animals were radiographically screened to ensure propersize, skeletal maturity, and that no obvious osseous abnormalitiesexist. In each group of four dogs, the left side defects receivedimproved osteogenic device. The right side defects receivedmatrix/binding agent in two animals, and the remaining two animals wereuntreated. At sacrifice, the distal femurs were retrieved en bloc, andthe defect sites evaluated histologically and grossly based on upon theabove-described scheme.

The improved osteogenic device comprises standard device (2.5 mgrhOP-1/1 g matrix) admixed with CMC. To formulate the improved device,100 mg of the rhOP-1/collagen mixture were admixed with 20 mg of CMCimmediately prior to implantation (total 120 mg). The collagen onlydevice consists of bovine Type I collagen (100 mg). The study design issummarized in Table 17.

TABLE 17 Dog Osteochondral Defect Repair Dogs Group (2 defects/animal)Left Implant Right Implant Duration I 4 OP-1/CMC None/Vehicle  6 weeksII 4 OP-1/CMC None/Vehicle 12 weeks III 4 OP-1/CMC None/Vehicle 26 weeksIV 4 OP-1/CMC None/Vehicle 52 weeks OP 1/CMC: 120 mg OP-1 CMC/CollagenDevice (improved osteogenic device) Vehicle: 100 mg CMC/Collagen.

Surgery

Using standard aseptic techniques, surgery was performed underisofluorane gas anesthesia. A medial parapatellar incision approximatelyfour centimeters in length was made. The patella was retracted laterallyto expose the femoral condyle. Using a ⅛ inch drill bit, a pilot holewas made in the weight bearing region of the medial femoral condyle. A 5mm drill bit with a specially designed sleeve to prevent over drillingof the defect depth (6 mm) was used to create the final defect. Aftercopious irrigation with saline to remove bone debris and spilled marrowcells, the appropriate experimental device was packed into the defectsite using a blunt probe. The joint capsule and soft-tissues were thenmeticulously closed in layers. The procedure was repeated on thecontralateral side with the appropriate implant.

Evaluation

Four animals each were sacrificed at 6 and 12 weeks and four animalswill be sacrificed at 26 and 53 weeks post-operative. Animals weresacrificed using an intravenous barbiturate overdose. The femurs wereimmediately harvested en bloc and stored in a saline soaked diaper. Highpower photographs of the defect sites were taken. Soft-tissues weremeticulously dissected away from the defect site. The proximal end ofthe femur was removed.

The gross appearance of the defect sites and repair tissue were gradedbased upon the above-described parameters by two independent observersblinded to the treatment assignment. Points were apportioned accordingto the presence of intra-articular adhesions, restoration of thearticular surface, cartilage erosion and appearance.

All specimens were prepared for histologic evaluation immediately aftergross grading and photography. The individual distal femurs were fixedby immersion in 10% buffered formalin solution or in 4% paraformaldehydesolution. On a water cooled diamond saw, each defect site was isolated.Three sections from three levels were cut from each block. Levels 1 and3 were closest to the defect perimeter. Level 2 was located at thedefect center. Three sections from each level were stained with eitherhematoxylin and eosin, Goldner's trichrome, Safranin O, or Fast Green.Sections were then graded based upon the above-described scheme. Thisanalysis apportioned points based upon the nature of the repair tissue,structural characteristics, and cellular changes. A total of 24 pointsare possible.

Result and Conclusion

After 6 weeks, certain of the above-treated animals were sacrificed andimmunohistochemical evaluations were conducted as described elsewhereherein. The results were as follows: In all cases, defects treated withOP-1 CMC/collagen device exhibited superior repair. With the OP-1CMC/collagen device, there was unexpectedly complete or nearly completebridging of the defect with cartilage tissue. Type II collagen stainingwas observed in the reparative cartilage with little or no Type Icollagen staining. Proteoglycan staining followed the type II collagenlocalization with darker staining in areas that more closely resembledmature hyaline cartilage. Based on Safranin-O staining, regeneration ofsurface layer of cartilage was not yet complete at 6 weekspost-treatment.

After 12 weeks, healing had significantly progressed in defects treatedwith improved devices. No appreciable healing was observed in thecontrols. The mean gross grading score observed with improved devices at12 weeks was 6.50±0.89 (n=8); control means was 3.69±0.70 (n=8). At allremaining time points, it is anticipated that defects treated with theimproved osteogenic devices will demonstrate more advanced cartilageregeneration, chondrocyte and cartilage phenotype, in an acceleratedmanner relative to defects treated with only collagen/CMC or leftuntreated. The defects treated with improved osteogenic device areanticipated to exhibit cartilage and subchondral bone tissue, whereasthe collagen/CMC treated or untreated defects are expected to inducedisorganized bone and cartilage formation with considerable fibroustissue present.

Full-thickness osteochondral defects can be stably repaired usingCMC-containing osteogenic devices in accordance with the methods of theinstant invention. It is expected that experiments similar to thosedescribed above, in which other preferred binding agents such as fibringlue are evaluated, will demonstrate that improved osteogenic devicescan stably repair full-thickness osteochondral defects.

E. Repair of Chondral Defects Using Improved Osteogenic DevicesContaining Carboxymethylcellulose

1. Experiment 1: Long Term Evaluation of Repair Chondral vs.Osteochondral Defects (Sheep)

This study evaluates repair of both chondral and osteochondral defectsby improved osteogenic devices using a large animal model. The increasedthickness of the articular cartilage and the similarities to humans insize and weight-bearing characteristics make the sheep a model fromwhich human clinical applications can be extrapolated, especially forclinical application of improved osteogenic devices for repair ofchondral defects. The study groups are as follows:

A-Osteochondral (full-thickness) Defects (5 mm diameter);

-   -   Group A I: no treatment    -   Group A II: carboxymethylcellullose/collagen    -   Group A III: carboxymethylcellullose/OP-1/collagen    -   Group A IV: lyophilized allograft    -   Group A V: lyophilized allograft+OP-1

B-Chondral (partial thickness) defects (5 mm diameter);

-   -   Group B I: no treatment    -   Group B II: carboxymethylcellullose/collagen    -   Group B III: carboxymethylcellullose/OP-1/collagen    -   Group B IV: hyaluronic acid+chondroitin sulfate paste    -   Group B V: hyaluronic acid+chondroitin sulfate paste+OP-1

Both foreknee joints of each sheep are operated on, and two defects perjoint are created (one each on the medial and the lateral condyle). Oneof the joints has two standardized partial thickness chondral defects (5mm in diameter) created on each condyle, while the other joint has twodeeper, full thickness osteochondral defects (about 1-2 mm into thesubchondral bone) created. Each group has a subgroup sacrificed early at8 weeks and another kept for longer term evaluation for 6-7 months.

There are a total of 20 groups and 12 defects per group. Therefore, thetotal number of defects is 240 and total number of sheep is 60. Thereare five different treatment groups; three controls (no treatment andtwo different mock devices) and two different OP-1 formulations for eachdefect type. Improved osteogenic devices comprising OP-1 in CMC/collagenwill be used for osteochondral defect repair and chondral defect repair.The devices are formulated such that 2.5 mg OP-1/g collagen are added toeach defect site receiving this improved osteogenic device. Repair isevaluated at 8 weeks and 6-7 months. The treatment protocol is shown inTable 18.

TABLE 18 Sheep Chondral and Osteochondral Defect Repair using OP-1 SheepOsteochondral (4 defects/ (2 defects/ Chondral Defects Group sheep)sheep) (2 defects/sheep) Duration I 12 Untreated Untreated Control    8weeks (6) Control >26 weeks (6) II 12 CMC/Collagen CMC/Collagen    8weeks (6) Control Control >26 weeks (6 III 12 OP-1 + OP-1 +    8 weeks(6) CMC/Collagen CMC/Collagen >26 weeks (6

Surgeries on the two knees are staggered by two weeks to allow healingof the first knee prior to surgery on the second knee. The first surgeryis used to generate chondral defects, the second is for osteochondraldefects. The surgery is performed in a fully equipped operating roomusing standard techniques and equipment used in human surgery. The sheepare allowed to ambulate freely in their pasture territorypost-operatively. Staggered surgeries result in 8 week healing times forchondral defects and 6 week healing times for osteochondral defects. Atsacrifice, the joints are perfused, fixed and processed according tostandard cytological protocols.

At the end of the study periods, the animals are sacrificed and thejoints are harvested en bloc. The gross appearance of the defect sitesand repair tissue is graded using routine methods such as thosedescribed above. Points are apportioned according to the presence ofintra-articular adhesions, restoration of articular surface, cartilageerosion and appearance.

Using methods similar to those described above, specimens are preparedfor histologic evaluation immediately after gross grading andphotography.

It is expected that defects treated with OP-1/CMC/collagen devices willexhibit superior repair similar to that in Experiment D.2 above. It isfurther expected that improved osteogenic devices containing any of theaforementioned preferred binding agents, such as fibrin glue, will alsoexhibit repair of both chondral and osteochondral defects.

2. Experiment 2: Long Term Evaluation of Repair of Using Varying DosesOf OP-1 Subchondral Defects (Goats)

A study using skeletally mature milk-goats is conducted to demonstratethe efficacy of improved osteogenic device for repairingosteochondral/chondral defects. Formulations of improved osteogenicdevice with varying concentrations of rhOP-1 are used, along with mockor no-device controls. The mock device consists of collagen admixed withcarboxymethylcellulose (CMC). Furthermore, the animal groups aresacrificed at 4, 12 and 24 months after surgery to compare the rate andstability of defect repair. The following summarizes the experimentalparameters.

Groups

Post-operation time: 4 mo. 12 mo. 2 year 1. rhOP-1 800 μg/ml A B C 2.rhOP-1 1600 μg/ml A 3. rhOP-1 3200 μg/ml A 4. Mock device A B 5. Nodevice A B

Briefly, subchondral defects are made in the left knees of 56 skeletallymature milk-goats: The defects are 8 mm in diameter and 3 mm in depth.This defect configuration prevents very high shear stresses in thedefect leading to collagen Type I formation. Dutch milk-goats, about 2years old and weighing approximately 50 kg are used in this experiment.Devices corresponding to 2.5 mg rhOP-1/gram collagen are provided. Ineach case, 0.2 grams of CMC are added to standard osteogenic device,then approximately 2.6 ml of saline are added and mixed. This yieldsmaterial of approximately 3-4 ml of improved osteogenic device. Thismaterial is then used to fill the defect volume.

Surgical Technique

Anesthesia is induced and the left knee is opened via a medialparapatellar approach. The patella is dislocated to the lateral side andthe medial condyle is exposed. With a sharp hollow tube, the outlines ofa defect are made in the anterior weight bearing part of the medialcondyle. With a square pointed handburr that is placed inside the tube,a defect down to the subchondral bone is created. The proximal tibia isthen exposed, and a periosteal flap of the same diameter as the defectin the medial condyle is taken. The periosteal flap is partially fixed,with its cambium layer towards the defect, to the remnants. The defectis filled with the appropriate test material and covered with theperiosteal flap, using a resorbable suture. The CMC device is added viaa syringe until the defect is filled, and the flap is then completelysutured. In control animals, a mock device including collagen and CMConly is used. A second control group received no implant at all, butreceived only a periosteal flap.

Post-Operative Treatment

Unrestricted, weight-bearing activity is allowed as much as can betolerated post-operatively.

Clinical Performance

The weight bearing pattern is assessed at 2, 4, 6, and 8 weeks, and thenevery 4 weeks.

Gross Analysis

Gross evaluations are made based upon the scheme presented above. Aftersacrificing the animal, the presence or absence of knee contractures isrecorded, and both the patella and condyles of the femur are examinedfor adhesions, articular surface contour, the appearance of the restoredcartilage, and the presence or absence of cartilage erosions. Each ofthese characteristics is given a score. Color slides are taken using amacro-lens.

Histological Analysis

To aid in visualization of the regenerated subchondral bone and tolocalize the borders of the defect during histological evaluation, thegoats receive a double labeled tetracycline before sacrificing. Thisallows histomorphometry of the bony filling of the deeper part of thedefect. The histological samples are also viewed by incorporatingpolarized microscopy to provide information on regular structuralfeatures.

For histological analysis, the specimens, including the subchondralbone, are fixed in 10% phosphate buffered formalin and are embeddedundecalcified in methylmethacrylate (MMA). With a heavy duty microtome,sections of 5 μm thick are made. The sections are stained with toluidineblue to identify cartilage and with Goldner's Trichrome to identifybone. Assessment is made of tissue hyalinity, affinity of the matrix fortoluidine blue (metachromasia), surface irregularity, chondrocyteclustering regenerated subchondral bone, bonding to the adjacentarticular cartilage, inflammatory cell infiltration around the implant,and freedom from degenerative changes in the adjacent cartilage. Each ofthese characteristics is given a score.

Biochemical Analysis

Extraction of proteoglycans: For biochemical analysis, control cartilageand tissue from the defect is collected in cold phospate-buffered saline(PBS). Proteoglycans are extracted from lyophilized sections bytreatment with 4 M guanidine HCl, 0.15 M potassium acetate at pH 5.8 inthe presence of proteinase inhibitors (5 mM benzamidine, 0.1 M6-amino-n-hexanoic acid, 10 mM EDTA, 5 mM phenylmethylsulfonyl fluoride,and 5 mM n-ethylmaleimide) at 4° C. for 60 hours. The extract andresidue are separated. The residue is thoroughly rinsed with extractionbuffer, which is added to the extract. The extracts are analyzed forchondroitinsulphate content and used for gel filtration.

Gel filtration: Aliquots of extracts are applied to Sepharose C 12 Bcolumns (0.66×145 cm) (Pharmacia AB, Uppsala, Sweden) and eluted with adissociative buffer at pH 6.1, containing 4 M guanidine HCl, 0.1 sodiumsulfate, 0.05 M sodium acetate, and 0.1% triton X-100. The flow rate is1.2 ml/hr. Fractions are analyzed for chondroitinsulphate content. Theamount of large cartilage-specific molecules, probably aggrecans, can becalculated.

MRI

Magnetic resonance imaging (MRI) is performed for 2 purposes. First, tomonitor 1 month post-operatively that the flap plus implant has remainedin place. Second, group 1C (2 years or more post-op) is followedlongitudinally with MRI at 4 months, 12 months and at sacrifice.

Summary

It is anticipated that defects treated with improved osteogenic devicewill demonstrate advanced cartilage regeneration, chondrocyte andcartilage phenotype compared to the mock or no-device controls. It isalso anticipated that low doses of OP-1 will at least achieve repairquantitatively and qualitatively similar to that of higher doses.

F. Repair of Chondral Defects Using Osteogenic Protein

This study investigated mammalian cartilage formation in subchondrallesions treated with recombinant human osteogenic protein-1 (rhOP-1)(alone or in combination with a collagen matrix) and/or autologousperichondrium.

Material and Methods

In the medial femoral condyle of the left knee joint of 15 goats, asubchondral defect of 9 mm diameter was made. The defect was filled withan implant consisting of fresh coagulated blood mixed with: (a) smallparticles of autologous ear perichondrium; or (b) rhOP-1; or (c) rhOP-1plus ear perichondrium. Rh—OP-1 was either added in combination with acollagen matrix (OP-1 Device) or without a collagen matrix (OP-1 alone).The defect was closed with a periosteal flap, which was stitched to thecartilage. After implantation times of 1, 2 and 4 months, the extent ofrepair of each defect was investigated with standard histologicaltechniques (metachromasia and hyalinity) and well-known biochemicalmethods (gel chromatography of proteoglycans).

Results

After 1 and 2 months in this particular study, there were no apparentdifferences between control (implant (a) above) and the various OP-1treated defects. However, after 4 months, only one out of three controldefects showed detectable cartilage formation, while all four OP-1treated defects were completely or partly filled with cartilage, asindicated by the histological and biochemical analysis set forth inTable 19.

TABLE 19 A B % of Biochemical Histology Cartilage Score² Implant defect¹Score Score partial total Control 86% 0.7 0.0 0.60 14% 3.0 4.0 0.98 1.58OP-1 62% 2.0 4.0 3.72 Device 38% 3.3 6.0 3.53 7.25 OP-1 79% 1.2 2.0 2.53Device + 21% 5.7 6.0 2.46 7.99 perichon. OP-1 79% 2.0 5.0 5.53 alone 21%2.1 6.0 1.70 4.23 OP-1 + 78% 1.0 2.0 2.34 perichon. 22% 4.2 5.0 2.024.36 ¹The defect was divided into homogeneous parts, the % is indicated.²Calculated as follows: % × (A + B), e.g. 0.86 × (0.7 + 0.0) = 0.60.

Table 19 sets forth the cartilage score of condylar defects, treated for4 months without OP-1 (control) or with OP-1 plus or minus perichondriumin the presence or absence of a collagen matrix.

Biochemical score (A) was assigned a value from 0-5 based on gelchromatography.

Histology score (B) is based on undecalcified plastic sections on agrading scale of 0 to 6.

Conclusion

The results of this study confirm that OP-1 has cartilage-promotingutility in large subchondral defects in goats. This indicates that OP-1is of clinical relevance in treating large lesions of articularcartilage and is particularly useful for chondral repair ofweight-bearing skeletal defects caused by trauma or disease in mammals.

In related studies, it is anticipated that other improved osteogenicdevices such as fibrin-glue containing devices, will result in repair oflarge subchondral defects. Moreover, such repair will be accompanied byregeneration of more stable, pristine articular cartilage. It is furtheranticipated that subchondral defect repair will occur at an acceleratedrate with reduced amounts of OP-1 when admixed with collagen matrix anda binding agent, such as CMC, relative to OP-1 admixed with collagenalone. Moreover, such repair will be accompanied by regeneration of morestable, pristine articular cartilage.

G. Segmental Defect Repair (Critical and Non-Critical Size) UsingImproved Osteogenic Devices Comprising Apatites and/or TriCalciumPhosphates (TCP) and/or Collagen Matrices

Improved devices comprising a variety of matrices or admixtures thereofwill be used to repair segmental ulna defects (critical and non-criticalsize) at varying doses of OP-1 in rabbits and dogs. Improved deviceswill comprise: Pyrost® matrix (Osteo AG, Switzerland), a HAp blockderived from bovine bone; 100% HAp granules (approximately 300-400 or350-450μ); 100% TCP (approximately 400μ); and 50% HAp/50% TCP(approximately 400μ). Other embodiments will comprise one or more of theearlier-described matrices of appropriate porosity. One particularlypreferred embodiment of improved osteogenic device will compriseCollapat® matrix (Osteo AG, Switzerland), a sponge of HAp and collagen.Another particularly preferred embodiment comprises approximately 0.6 gCMC per g HAp granules or per g granules of 75% HAp/25% TCP, especiallywhen a device with putty consistency is desired. Another preferredembodiment described above contains β-TCP and fibrin glue.

It is expected that improved devices such as those described above willinduce repair of segmental defects, and certain preferred embodimentswill do so at low doses of OP-1.

H. Bone Formation Using Fibrin Glue as Binding Agent

Four rat subcutaneous studies were completed for evaluating fibrin glueOP-1 formulation on bone formation. The amount of bone formation at 10μg OP-1 using the three different sources of fibrin glue were similar,ranging from 25% to 40% (See Tables 19A-19F). There was no clearcorrelation between inflammation and bone formation in these studies.Results indicated that rat reacted differently to fibrin glue fromdifferent species; For example, human fibrin glue from Tissucol®elicited an inflammation response from 2 to 2.7 (See Table 19A), bovinefibrin glue caused an inflammation response from 2 to 3.5 (See Tables19B and 19C) and rat fibrin glue had the lowest inflammation responsefrom 1 to 1.3 (See Table 19D) on a scale of 0-4. Typically, aninflammation response of 3-4 is defined as severe and 1-2 is defined asmild to medium.

TABLE 19A IN VIVO data of Tissucol ®/OP-1 OP-1, μg Half Explant % Bone/Fibrosis Inflammation Study n = 4 wt, mg Ca⁺², μg/mg Histology (0-4)(0-4) Tissucol ® 0 n.d. <1 0 2.3 +/− 0.6 2.7 +/− 1.2 Tissucol ® 10 16+/− 6   15 +/− 23 25 +/− 25 3.3 +/− 1.0 2.5 +/− 0.6 Tissucol ® 20 58 +/−42 39 +/− 9 66 +/− 32 1.8 +/− 0.5   2 +/− 0.8 OP-1 10 29 +/− 21 49 +/− 895 +/− 6  0.8 +/− 0.5 0.5 +/− 0.6 Twenty μL OP-1 (10 μg or 20 μg in 5%lactose) was mixed with 50 μL fibrinogen (70-110 mg/mL) and 50 μLthrombin solution (500 U/mL) prior to subcutaneous implantation.

TABLE 19B IN VIVO data of Bovine Fibrin Glue OP-1, Half μg Explant wt, %Bone/ Fibrosis Inflammation (Implant) n = 4 mg Ca⁺², μg/mg Histology(0-4) (0-4) 6% fibrin 0 n.d. glue 6% fibrin 10 18 +/− 8 25 +/− 12 40 +/−43 2.3 +/− 0.6 2 +/− 1 glue OP-1 10 58 +/− 8 65 +/− 10 100 +/− 0  0.75+/− 0.5  0 +/− 0 Twenty μL OP-1 (10 μg in 5% lactose) was mixed with 50μL bovine fibrinogen (60 mg/mL) and 50 μL bovine thrombin solution (300U/mL) prior to subcutaneous implantation.

TABLE 19C IN VIVO data of Bovine Fibrin Glue OP-1, μg Half Explant Ca⁺²,% Bone/ Fibrosis Inflammation (Formulation) n = 4 wt, mg μg/mg Histology(0-4) (0-4) 4% fibrin glue 0 n.d. <3 0 2.25 +/− 1     3 +/− 1.4 4%fibrin glue 10 15 +/− 7  10 +/− 8 24 +/− 25 2.5 +/− 0.6 3.5 +/− 1   OP-110 8 +/− 5 42 +/− 7 88 +/− 10 1 +/− 0 0 +/− 0 Twenty μL OP-1 (10 μg in5% lactose) was mixed with 50 μL bovine fibrinogen (40 mg/mL) and 50 μLbovine thrombin solution (200 U/mL) prior to subcutaneous implantation.

TABLE 19D IN VIVO data of Rat Fibrin Glue OP-1, Half μg Explant, Ca⁺², %Bone/ Fibrosis Inflammation n = 4 mg μg/mg Histology (0-4) (0-4) % CystRat fibrin 10 32 +/− 13 2 +/− 0.6  13 +/− 19 1.3 +/− 0.5 1.0 +/− 0.8  9+/− 12 dil. 2 10 38 +/− 23 2 +/− −0.8 23 +/− 21 1.8 +/− 0.5 1.3 +/− 0.526 +/− 18 Twenty μL OP-1 (10 μg in 5% lactose) was mixed with 50 μLbovine fibrinogen (40 mg/mL) and 50 μL bovine thrombin solution (200U/mL) prior to subcutaneous implantation.

Two other rat in vivo studies were completed for evaluating devicescontaining fibrin glue and OP-1 on bone formation. In the first study,bovine fibrinogen (504, Sigma F8630, 10 mg/mL) and bovine thrombin (50μL, 50 U/mL) were mixed with OP-1 devices right before subcutaneousimplantation. The positive controls are OP-1 devices wetted with 100 μLphosphate buffered saline. The OP-1 devices were prepared by mixing 10μg OP-1 in 47.5% ethanol/0.01% TFA with 25 mg collagen and lyophilizedovernight. Results are shown in TABLE 19E. There was no significantdifference in bone formation between standard OP-1 devices and OP-1devices combined with bovine fibrin glue. Also, a lower inflammationresponse was observed to the OP-1 devices/bovine fibrin glue formulationcompared with the combination of liquid OP-1 bovine fibrin glue (seeTABLES 19B and 19C).

TABLE 19E IN VIVO data of Bovine Fibrin Glue with OP-1 Device OP-1, Halfμg Explant, % Bone/ Fibrosis Inflammation n = 4 mg Ca⁺², μg/mg Histology(0-4) (0-4) Fibrin + OP- 0  62 +/− 15 20 +/− 7 0 +/− 0 2.0 +/− 0.0 1.0+/− 0.0 1 device Fibrin + OP- 10 123 +/− 21  43 +/− −11 66 +/− 13 1.5+/− 0.6 1.3 +/− 0.5 1 device OP-1 device 10 144 +/− 33 54 +/− 5 78 +/−13 1.0 +/− 0.8 0.5 +/− 0.6

In a second study, different concentrations of Tissucol® were combinedwith the standard OP-1 device. That is, one lyophilized OP-1 device (25mg total weight, 10 μg OP-1) was combined with human fibrinogen solution(50 pit, fibrinogen 70-110 mg/mL or diluted 2 or 4 or 8 folds inphosphate buffered saline) and thrombin solution (50 μL, 500 U/mL ordiluted 2 or 4 or 8 folds in phosphate buffered saline) immediatelyprior to implantation. The positive controls are OP-1 devices wettedwith 100 μL phosphate buffered saline. Results are shown in TABLE 19F.There is no significant difference in bone formation between standardOP-1 devices and OP-1 devices combined with different concentration ofTissucol®. Also, the concentration of fibrinogen had no significanteffect on bone formation.

TABLE 19F IN VIVO data of Tissucol ® with OP-1 Devices OP- 97-0137 1, μgHalf % Bone/ Fibrosis Inflammation (#1111) n = 4 Explant, mg Ca⁺², μg/mgHistology (0-4) (0-4) OP-1 + 10 163 +/− 50 37 +/− 7 55 +/− 30 2.0 +/−0.8 1.8 +/− 1.0 Tissucol ® OP-1 + 2 10 162 +/− 32 34 +/− 8 54 +/− 13 1.8+/− 1.0 1.8 +/− 1.0 folds diluted Tissucol ® OP-1 + 4 10 170 +/− 19 41+/− 6 69 +/− 26 1.5 +/− 1.0 1.5 +/− 1.0 folds diluted Tissucol ® OP-1 +8 10 177 +/− 23 40 +/− 8 55 +/− 25 1.8 +/− 1.0 1.8 +/− 1.0 folds dilutedTissucol ® OP-1 device 10 137 +/− 43  58 +/− 10 84 +/− 8  1.0 +/− 0.01.0 +/− 0.0

In summary, the handling property of standard. OP-1 devices is improvedby using fibrin glue, e.g., and the collagen particles remain integratedwith the glue. The in vivo data indicated that OP-1 devices with fibringlue promote bone formation.

I. Defect Repair Using Improved Osteogenic Devices Containing FibrinGlue as Binding Agent

Fibrin glue-containing improved devices comprising a variety of matricesor admixtures thereof will be used to repair bone, osteochondral orchondral defects at varying doses of OP-1 art-recognized animal models.Certain embodiments of preferred devices will comprise: fibrin glue,collagen and OP-1. Other embodiments of preferred devices will comprise:fibrin glue, β-TCP and OP-1. Finally, further testing will include anyof the aforementioned matrix materials suitable for use with the presentinvention.

It is expected that fibrin glue-containing improved devices such asthose described above will promote and accelerate bone formation incritical and non-critical sized defects, non-union fractures, andfractures, as well as promote and accelerate repair of osteochondral andchondral defects.

J. Segmental Defect Repair (Critical and Non-Critical Size) Using FibrinGlue-Containing Improved Device

The following is a comparative experimental study of the efficacy offibrin glue-containing improved devices for healing segmental (criticaland non-critical sized) defects.

Test System

As described above, adult male mongrel dogs bred for purpose areutilized in this study. Special attention is paid in selecting animalsof uniform size and weight to limit the variability in bone geometry andloading.

As also described above surgery is performed using standard aseptictechniques under isofluorane gas anesthesia. Both forelimbs are preppedand draped in sterile fashion. A lateral incision approximately twocentimeters in length is made and exposure of the ulna is obtained usingblunt and sharp dissection. Either a critical or a noncritical sizeddefect is created in the mid-ulna using an oscillating saw. The radiusis maintained for mechanical stability and no internal or externalfixation is used. The site is irrigated with saline and the soft tissuesmeticulously closed in layers around the defect. The appropriate implantdevice is implanted or injected into the defect site. The procedure isthen repeated on the contralateral side with the appropriate implant.

Animals are administered intramuscular antibiotics for four dayspost-surgery and routine anterior-posterior radiographs are takenimmediately after surgery to insure proper surgical placement. Animalsare kept in 3×4 foot recovery cages until weight bearing isdemonstrated, after which they are transferred to runs and allowedunrestricted motion.

Radiographs of the forelimbs are obtained weekly until four weeks, andthen biweekly to 16 weeks in surviving animals using standardizedexposure times and intensities. Radiographs are evaluated and comparedto earlier radiographs to appreciate quality and speed of defecthealing. Changes in radiographic appearance are evaluated based onpresence and density of new bone formation, extent of defect bridgingand incorporation of the host bone cortices.

Test Material Description

The implant materials contain recombinant human osteogenic protein-1(rhOP-1) in an acetate buffer formulation, and rhOP-1 in either fibringlue plus collagen or fibrin glue plus β-TCP. The rhOP-1 formulationsare compared to vehicle only controls. The acetate buffer rhOP-1formulation consists of 3.5 mg/ml OP-1 in a lactose/acetate bufferdelivered in a 100 μl volume. The vehicle control consists of a 100 μlvolume of lactose/acetate buffer. Test formulations containrhOP-1/fibrin glue-collagen or rhOP-1/fibrin glue β-TCP.

Experimental Design

Bilateral ulna segmental defects, critical or non-critical size, arecreated in all animals. One group of animals receive an injection of0.35 mg rhOP-1/acetate buffer formulation in one defect and the acetatebuffer without rhOP-1 in the contralateral defect. Another group ofanimals receive an injection of rhOP-1/fibrin glue-collagen orrhOP-1/fibrin glue-β-TCP formation in one defect and fibrin glue-β-TCPor collagen alone in the contralateral defect. Animals are sacrificed atperiods of 4, 8 and 12 weeks postoperative. Certain dogs receivebilateral defects with no implant (defect only) and are evaluated atperiods of 4, 8, 12 and 16 weeks postoperative.

Testing Procedures

At the end of the study period, animals are sacrificed using anintravenous barbiturate overdose. The ulna and radius are immediatelyharvested en bloc and placed in saline soaked diapers. Both ulna aremacrophotographed and contact radiographs taken before soft tissues werecarefully dissected away from the defect site. A water-cooled saw isthen used to cut the ulna to a uniform length of 9 cm with the defectcentered in the middle of the test specimen for biomechanical testingevaluation.

If defect healing is sufficient based upon manual manipulation,specimens are tested to failure in torsion on an MTS closed-loophydraulic test machine (Minneapolis, Minn.) operated in stroke controlat a constant displacement rate of 50 mm/min. Each end of the bonesegment is mounted in a cylindrical aluminum sleeve and cemented withmethylmethacrylate. One end is rigidly fixed and the other is rotatedcounterclockwise. Since the dog ulna has a slight curvature, thespecimens are mounted eccentrically to keep specimen rotation coaxialwith that of the testing device. The torsional force is applied with alever arm of 6 cm. Force-angular displacement curves are generated fromwhich the torque and angular deformation to failure are obtained, andthe energy absorption to failure is computed as the area until theload-displacement curve.

Both tested and untested specimens are prepared for histologicevaluation. The individual specimens are fixed by immersion in 10%buffered formalin solution immediately following mechanical testing orafter sectioning in untested specimens. On a water-cooled diamond sawthe specimens are divided by bisecting the specimen down its long axis.This procedure results in two portions of each specimen for histologicpreparations, including undecalcified ground sectioning andundecalcified microtome sectioning. The histologic sections areevaluated for the quality of union, the appearance and quality of thecortical and cancellous bone, and bone remodeling.

Results

It is expected that fibrin glue-containing improved devices with any ofthe aforementioned preferred matrices, such as collagen or β-TCP, willpromote repair of both critical and non-critical size segmental defects.

VII. Human Clinical Studies: Methods of Use of Improved OsteogenicDevices A. Repair of Bone Defects.

1. Trial 1: Fresh Open Tibial Fracture.

This study is a multi-center, prospective, randomized study of patientswith fresh, fractures of the tibia requiring surgical intervention atthe fracture site.

Introduction

Currently there are approximately 26 million fractures annually worldwide. The majority of fractures heal without complication and are notconsidered “a problem”. There is a “quality of life impact,” however,with patients being out of work or prevented from engaging in normalactivity, not being able to return to activity or, when they do,suffering with lingering pain. Patients, particularly in the westernworld, are growing to expect solutions to these problems.

The cost of fracture treatment is astounding. In 1988, fractures cost anestimated $20 billion in the United States. The largest segment,approximately 44% or $7.2 billion, was related to in-patient treatment.In that year, almost 900,000 persons were hospitalized for fractures,with an average length of stay of 8.8 days for a total of 7.9 milliondays. Nursing home costs were second at $2.8 billion, and out-patienthospital care third at $1.8 billion.

When the economic ramifications of fractures are considered inconjunction with the quality of life impact, there is indeed a need forimprovement in treatment methodologies, especially fractures that areconsidered potentially problematic. These are fractures that because ofthe nature of the injury or mitigating host issues, may requireadditional surgical interventions, take an extended time to heal, and/ormay prevent full functional recovery.

Thus the study described below is designed to investigate improvedosteogenic devices as a healing accelerator for fresh fractures inhumans and as a means to decrease the potential for post-injury problemhealing requirement intervention to augment the healing process.Additionally, certain patients within this study will be treated withimproved osteogenic devices as a bone graft substitute in patientsrequiring bone grafting post-injury or in cases of delayed healing.

As contemplated herein and described above, currently preferredembodiments of improved osteogenic devices have a consistency which canbe injected through a large gauge needle or can be placed through anopen incision such that it will remain generally in place in a bloodyenvironment. In addition to more conventional packaging, the injectableimproved osteogenic devices can be packaged in applicator/syringe readyto be used. A variety of nozzles and needles can be added to customizeapplication. Other embodiments will also be rendered radio opaque byaddition of radio opaque components such as described earlier.

It is expected that the improved osteogenic devices of the instantinvention (injectable and implantable) will decrease the incidence ofadditional interventions, speed rate of healing, improve quality of lifeand speed return to normal activity. Furthermore, it is expected thatthe improved osteogenic devices disclosed herein will be used infractures of all long bones, clavicle, and scapula to promote healing,leading to decreased incidence of intervention (including re-operation),increased speed of healing, increased rate of return to normal activity,and decreased morbidity. In contrast to currently available fracturerepair modalities, there is no biomechanical requirement with theimproved devices and methods disclosed herein.

Study Design

Patients will require surgical treatment of open fractures of the tibiaacquired secondary to trauma. The fracture must have the potential to beadequately stabilized at the fracture site to permit healing. Patientswill show radiographic evidence of skeletal maturity.

Type of Treatment

-   -   Type #1: Initial injury at ≦7 days at definitive closure.    -   Type #2 At up to 6 weeks post-initial injury in patients        requiring bone grafting.

Type #1 fractures are those not requiring bone grafting. Patients willbe randomized in a 1:1 ratio of standard treatment (debridement offracture site, reduction and stabilization), which will be the controlgroup versus standard treatment plus OP-1 device with and without abinding agent, such as carboxymethylcellulose (CMC). In certainpatients, dosages of osteogenic protein OP-1 will vary. As describedabove, a currently preferred formulation of the improved osteogenicdevice contains 2.5 mg OP-1/1000 g collagen/200 mg CMC. OP-1 dosageswill vary from ½ maximal to 4×; CMC content will vary from 100-300 mg.As also described above, variations of wetting agent volumes will beinvestigated by the attending surgeon/physician to achieve the desiredconsistency/configuration of device. Patients from the first group whoare not healed 6 months post-treatment will again be randomized in a 1:1ratio of bone grafting (control) versus OP-1.

Type #2 fractures are those requiring bone grafting. Patients will berandomized in a 1:1 ratio of bone grafting (control) versus OP-1.Patients from the first group who are not healed 6 months post-treatmentwill be crossed over from bone grafting to OP-1 and from OP-1 to bonegrafting.

Study Plan

Patients will be followed for a minimum of 1 year post-treatment toassess healing, with a 24 month follow-up to assess status.

Follow-up assessments will be performed at 2 weeks, 4 weeks and every 4weeks up to 6 months, and at 8, 10 and 12 months post-treatment. Allpatients will have an additional follow-up assessment at 24 months todetermine overall health and fracture site status. The followingassessments will be performed: changes in physical examination;radiographs; clinical pain assessments; clinical assessments ofweight-bearing; clinical assessments of function; quality of lifeassessment (pre-discharge, 6 and 12 months); and documentation of anyinterventions to augment/promote healing (surgical and nonsurgical) andhardware failures/replacements.

It is expected that fractures treated with improved osteogenics willevidence an accelerated rate of healing.

Additionally, it is expected that the patients treated with improvedosteogenic devices will experience at least the following additionalbenefits:

-   -   1) Potential for decreased healing time with faster restoration        of function, weight bearing and ambulation;    -   2) Potential for prevention of delayed/mal/non-union;    -   3) Return to normal activities sooner/less time lost from        jobs/school;    -   4) Potential saving from further intervention/surgical        procedures for promotion of healing;    -   5) Less hardware complications; and    -   6) In those patients who require bond grafting, the benefit of        no second site surgery for bone harvest with associated        morbidity.

2. Trial 2; Fresh Closed Diaphyseal Fracture.

Repair of fresh closed diaphyseal fractures in human subjects will beevaluated using improved osteogenic devices. Specifically, patients willbe treated with injectable improved osteogenic devices by injecting thedevice at the closed defect site. It is anticipated that acceleratedrepair of the defect will be observed relative to patients not treatedwith improved osteogenic devices.

3. Other Human Trials

It is anticipated that Trials 1 and 2 as set forth above will berepeated using various configurations of improved osteogenic devicescontaining fibrin glue. It is further anticipated that such devices willpromote bone formation, and in certain embodiments accelerate defectrepair relative to untreated subjects.

B. Repair of Osteochondral Defects.

1. Experiment 1: Osteochondritis Dessicans

Osteochondral defect models support the clinical use of rhOP-1 to treatOsteochondritis Dissecans (OD) and trauma defects. OD is a diseaseresulting in localized areas of osteochondral defects. One cause of thedisease may be ischemia damage to the localized area, but its exactetiology is unknown. In patients with OD, the affected area becomesavascular, with subsequent changes in the overlying articular cartilage.Patient's suffering from OD of the knee experience symptoms includinglocking of the joint, localized pain, swelling and retropatellarcrepitus. An experiment involving patients with OD of the knee isconducted in order to compare the ability of improved osteogenic device,against that of standard osteogenic device, to repair OD defects.

Current methods known in the art for the treatment of OD involve the useof highly invasive surgical techniques. In most skelatally maturepatients with OD, surgery is required. Surgical techniques requirearthroscopic drilling of the intact lesion. As a result, patients mustundergo administration of general anesthesia during surgery.Post-operatively, patients must have movement of their knees restrictedby an immobilizing brace and cannot walk without the use of crutchesuntil healing is evidenced.

In this study, less invasive techniques for treatment of OD areconducted. The techniques involve the use of improved osteogenic device,which is delivered to the defect site via injection. The activity ofimproved osteogenic device in repair of OD is compared to that ofstandard osteogenic device.

It is anticipated that patients treated with the improved osteogenicdevice containing any of the aforementioned matrices and binding agentswill show greater relief of symptoms of OD than those treated withstandard osteogenic device. Patients treated with improved osteogenicdevice will experience at least a greater decrease in pain, swelling andlocking of the knee than those treated with standard osteogenic device,all of which are indicia of amelioration and/or repair of the defect.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are therefore intended to be embracedtherein.

What is claimed is:
 1. A device for inducing local bone or cartilageformation, comprising: osteogenic protein; matrix derived fromnon-synthetic, non-polymeric material; and binding agent.
 2. The deviceof claim 1, wherein said osteogenic protein is selected from the groupconsisting of: OP-1, OP-2, OP-3, BMP3, BMP4, BMP5, BMP6, BMP9, BMP10,BMP11, BMP12, BMP15, BMP16. DPP, Vg1, Vgr, 60A protein, GDF1, GDF3,GDF5, GDF6, GDF7, GDF8, GDF9, GDF10, GDF11, and amino acid sequencevariants of each of the foregoing.
 3. The device of claim 1, whereinsaid osteogenic protein is selected from the group consisting of: OP-1,OP-2, BMP2, BMP4, BMP5, BMP6, and amino acid sequence variants of eachof the foregoing.
 4. The device of claim 1, wherein said osteogenicprotein comprises an amino acid sequence having at least 70% homologywith the C-terminal 102-106 amino acids, including the conserved sevencysteine domain, of human OP-1.
 5. The device of claim 1, wherein saidosteogenic protein is OP-1.
 6. The device of claim 1, wherein saiddevice comprises at least two different osteogenic proteins.
 7. Thedevice of claim 1, wherein said matrix is selected from the groupconsisting of: collagen, demineralized bone, apatites, hydroxyapatites,tricalcium phosphates, and admixtures thereof.
 8. The device of claim 1,wherein said matrix is collagen.
 9. The device of claim 1, wherein saidmatrix is β-tricalcium phosphate.
 10. The device of claim 1, whereinsaid device comprises at least two different matrix materials.
 11. Thedevice of claim 1, wherein said binding agent is selected from the groupconsisting of mannitol, dextrans, white petrolatum, mannitol/dextrancombinations, mannitol/white petrolatum combinations, sesame oil, alkylcelluloses, fibrin glue, and admixtures thereof.
 12. The device of claim1, wherein said binding agent is selected from the group consisting ofalkylcelluloses.
 13. The device of claim 1, wherein said binding agentis selected from the group consisting of methylcellulose,methylhydroxyethylcellulose, hydroxyethylcellulose,hydroxypropylmethylcellulose, carboxymethylcellulose, sodiumcarboxymethylcellulose, hydroxyalkylcelluloses, and admixtures thereof.14. The device of claim 1, wherein said binding agent iscarboxymethylcellulose or the sodium salt thereof.
 15. The device ofclaim 1 wherein said binding agent is fibrin glue.
 16. The device ofclaim 15, wherein said fibrin glue is a mixture of mammalian fibrinogenand thrombin.
 17. The device of claim 1, wherein said device comprisesat least two different binding agents.
 18. The device of claim 1,further comprising a wetting agent.
 19. The device of claim 18, whereinsaid wetting agent is saline.
 20. A device for inducing local bone orcartilage formation comprising: osteogenic protein OP-1, approximately1.25 mg; collagen matrix, approximately 1000 mg; andcarboxymethylcellulose approximately at least 180 mg.
 21. The device ofclaim 20 further comprising OP-1, approximately at least 2.5 mg.
 22. Thedevice of claim 20 or 21, further comprising carboxymethylcellulose,approximately at least 200 mg.
 23. A device for inducing local bone orcartilage formation, comprising: osteogenic protein, approximately 0.4mg; collagen matrix, approximately 1000 mg; and fibrin glue,approximately at least 20 mg.
 24. The device of claim 23, furthercomprising fibrin glue, approximately 40 mg.
 25. A device for inducinglocal bone or cartilage formation, comprising: osteogenic protein,approximately 1.2 mg; β-tricalcium phosphate, approximately 1000 mg; andfibrin glue, approximately at least 40 mg.
 26. The device of claim 23 or25, further comprising fibrin glue, approximately 220 mg.
 27. The deviceof claim 23 or 25, wherein said fibrin glue is a mixture of mammalianfibrinogen and thrombin.
 28. The device of claim 27, wherein thethrombin content is between about 2.0 U and about 25 U.
 29. The deviceof claim 27, wherein the thrombin content is between about 5.0 U andabout 25 U.
 30. The device of claim 27 wherein the thrombin content isbetween about 2.5 U and about 5.0 U.
 31. The device of claim 27 furthercomprising a fibrinogen content of between about 20 mg and about 180 mg.32. A device for inducing local cartilage or bone formation, comprisingosteogenic protein and a carrier, wherein said carrier comprises onepart (w/w) binding agent to 50 parts (w/w) matrix.
 33. The device ofclaim 32, wherein said carrier comprises one part (w/w) binding agent to25 parts (w/w) matrix.
 34. The device of claim 32, wherein said carriercomprises one part (w/w) binding agent to 10 parts (w/w) matrix.
 35. Thedevice of claim 32 wherein said carrier comprises one part (w/w) bindingagent to five parts (w/w) matrix.
 36. The device of claim 32, whereinsaid earlier comprises less than five parts (w/w) matrix.
 37. A devicefor inducing local bone or cartilage formation, comprising osteogenicprotein and a carrier, wherein said carrier comprises 10 parts (w/w)binding agent to one part (w/w) matrix.
 38. The device of claim 37,wherein said carrier comprises less than 10 parts (w/w) binding agent.39. The device of claim 20, 23, 26, 32 or 37 further comprising saline.40. A method for inducing local bone or cartilage formation for repairof bone, cartilage or osteochondral defects, comprising the step of:providing a device of claim 1, 20, 23, 26, 32 or 37 to a defect site.41. The method of claim 40, wherein bone formation is endochondral boneformation.
 42. The method of claim 40, wherein cartilage formation isarticular cartilage formation.
 43. The method of claim 40, wherein saiddefect site is selected from the group consisting of: critical sizedefect, noncritical size defect, segmental non-union defect, nonunionfracture, fracture, osteochondral defect, and subchondral defect. 44.The method of claim 40, wherein the volume of osteogenic device providedto the defect site is sufficient to fill the defect site.
 45. A devicefor inducing local bone or cartilage formation, comprising: osteogenicprotein OP-1; collagen matrix; and carboxymethylcellulose.
 46. A devicefor inducing local bone or cartilage formation, comprising: osteogenicprotein OP-1; collagen matrix; and fibrin glue.
 47. A kit for inducinglocal bone or cartilage formation using the device of claim 1, the kitcomprising: (a) a first receptacle adapted to house an osteogenicprotein and a matrix material; and (b) a second receptacle adapted tohouse a binding agent, wherein the osteogenic protein and matrixmaterial are provided in the first receptacle of part (a), and thebinding agent is provided in the second receptacle of part (b).
 48. Thekit of claim 48, further comprising a third receptacle adapted to housea wetting agent.
 49. The kit of claim 48 wherein said first receptacleand said second receptacle are the same.